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Abstract:

The present specification discloses TEMs, compositions comprising such
TEMs, compositions comprising such TEMs and Clostridial toxins, methods
of treating a neuroendocrine disorder in an individual using such
compositions, use of such TEMs in manufacturing a medicament for treating
a neuroendocrine disorder, use of such TEMs and Clostridial toxins in
manufacturing a medicament for treating neuroendocrine disorder, use of
such TEMs in treating a neuroendocrine disorder, and use of such TEMs and
Clostridial toxins in treating a neuroendocrine disorder.

Claims:

1. A method of treating a neuroendocrine disorder in an individual, the
method comprising the step of administering to the individual in need
thereof a therapeutically effective amount of a composition including a
TEM comprising a targeting domain, a Clostridial toxin translocation
domain and a Clostridial toxin enzymatic domain, wherein the targeting
domain is a sensory neuron targeting domain, a sympathetic neuron
targeting domain, or a parasympathetic neuron targeting domain, and
wherein administration of the composition reduces a symptom of the
neuroendocrine disorder, thereby treating the individual.

5. The method of claim 1, wherein the TEM is administered to an Arnold's
nerve or a nerve from the recurrent laryngeal nerve complex.

6. A method of treating a neuroendocrine disorder in an individual, the
method comprising the step of administering to the individual in need
thereof a therapeutically effective amount of a composition including a
TEM comprising a targeting domain, a Clostridial toxin translocation
domain, a Clostridial toxin enzymatic domain, and an exogenous protease
cleavage site, wherein the targeting domain is a sensory neuron targeting
domain, a sympathetic neuron targeting domain, or a parasympathetic
neuron targeting domain, and wherein administration of the composition
reduces a symptom of the neuroendocrine disorder, thereby treating the
individual.

13. The method of claim 6, wherein the TEM is administered to an Arnold's
nerve or a nerve from the recurrent laryngeal nerve complex.

14. A use of a TEM in the manufacturing a medicament for treating a
neuroendocrine disorder in an individual in need thereof, wherein the TEM
comprising a targeting domain, a Clostridial toxin translocation domain
and a Clostridial toxin enzymatic domain, wherein the targeting domain is
a sensory neuron targeting domain, a sympathetic neuron targeting domain,
or a parasympathetic neuron targeting domain.

[0003] Clostridial toxin therapies have been successfully used for many
indications. However, toxin administration in some applications can be
challenging because of the larger doses required to achieve a beneficial
effect. Larger doses can increase the likelihood that the toxin may move
through the interstitial fluids and the circulatory systems, such as,
e.g., the cardiovascular system and the lymphatic system, of the body,
resulting in the undesirable dispersal of the toxin to areas not targeted
for toxin treatment. Such dispersal can lead to undesirable side effects,
such as, e.g., inhibition of neurotransmitter release in neurons not
targeted for treatment or paralysis of a muscle not targeted for
treatment. For example, a individual administered a therapeutically
effective amount of a BoNT/A treatment into the neck muscles for cervical
dystonia may develop dysphagia because of dispersal of the toxin into the
oropharynx. As another example, a individual administered a
therapeutically effective amount of a BoNT/A treatment into the bladder
for overactive bladder may develop dry mouth and/or dry eyes. Thus, there
still remains a need for treatments having the therapeutic effects that
only larger doses of a Clostridial toxin can currently provide, but
reduce or prevent the undesirable side-effects associated with larger
doses of a Clostridial toxin administration.

[0004] A Clostridial toxin treatment inhibits neurotransmitter release by
disrupting the exocytotic process used to secret the neurotransmitter
into the synaptic cleft. There is a great desire by the pharmaceutical
industry to expand the use of Clostridial toxin therapies beyond its
current myo-relaxant applications to treat sensory, sympathetic, and/or
parasympathetic nerve-based ailments, such as, e.g., various kinds of
smooth muscle-based disorders. One approach that is currently being
exploited involves modifying a Clostridial toxin such that the modified
toxin has an altered cell targeting capability for a neuronal or
non-neuronal cell of interest. Called re-targeted endopeptidases or
Targeted Vesicular Exocytosis Modulator Proteins (TVEMPs) or Targeted
Exocytosis Modulators (TEMs), these molecules achieve their exocytosis
inhibitory effects by targeting a receptor present on the neuronal or
non-neuronal target cell of interest. This re-targeted capability is
achieved by replacing the naturally-occurring binding domain of a
Clostridial toxin with a targeting domain showing a selective binding
activity for a non-Clostridial toxin receptor present in a cell of
interest. Such modifications to the binding domain result in a molecule
that is able to selectively bind to a non-Clostridial toxin receptor
present on the target cell. A re-targeted endopeptidase can bind to a
target receptor, translocate into the cytoplasm, and exert its
proteolytic effect on the SNARE complex of the neuronal or non-neuronal
target cell of interest.

[0005] The present specification discloses TEMs, compositions comprising
TEMs, and methods for treating an individual suffering from a smooth
muscle-based disorder. This is accomplished by administering a
therapeutically effective amount of a composition comprising a TEM to an
individual in need thereof. The disclosed methods provide a safe,
inexpensive, out patient-based treatment for the treatment of involuntary
movement disorders. In addition, the therapies disclosed herein reduce or
prevent unwanted side-effects associated with larger Clostridial toxin
doses. These and related advantages are useful for various clinical
applications, such as, e.g., the treatment of smooth muscle-based
disorders where a larger amount of a Clostridial toxin to an individual
could produce a beneficial effect, but for the undesirable side-effects.

SUMMARY

[0006] With reference to neuroendocrine disorders as disclosed herein, and
without wishing to be limited by any particular theory, it is believed
that sympathetic, parasympathetic, and/or sensory neurons have important
functions in aspects of neuroendocrine regulation and that improper
innervations from these types of neurons can contribute to one or more
different types of neuroendocrine disorders. As such, TEMs comprising a
targeting domain for a receptor present on sympathetic, parasympathetic,
and/or sensory neurons can reduce or prevent these improper innervations,
thereby reducing or preventing one or more symptoms associate with a
neuroendocrine disorder. It is further theorized that such a TEM in
combination with a Clostridial toxin can provide enhanced, if not
synergistic, therapeutic benefit because such a combination also inhibit
motor neurons. However, using a combination therapy of such a TEM with a
Clostridial toxin, also allows a lower dose of a Clostridial toxin to be
administered to treat a neuroendocrine disorder. This will result in a
decrease in muscle weakness generated in the compensatory muscles
relative to the current treatment paradigm. As such, a combined therapy
using a Clostridial toxin and a TEM comprising a targeting domain for a
receptor present on sympathetic, parasympathetic, and/or sensory neurons
can reduce or prevent these improper innervations, and in combination can
reduce or prevent one or more symptoms associate with a neuroendocrine
disorder.

[0007] Thus, aspects of the present specification disclose methods of
treating a neuroendocrine disorder in an individual, the methods
comprising the step of administering to the individual in need thereof a
therapeutically effective amount of a composition including a TEM,
wherein administration of the composition reduces a symptom of the
neuroendocrine disorder, thereby treating the individual. In some
aspects, a TEM may comprise a targeting domain, a Clostridial toxin
translocation domain and a Clostridial toxin enzymatic domain. In some
aspects, a TEM may comprise a targeting domain, a Clostridial toxin
translocation domain, a Clostridial toxin enzymatic domain, and an
exogenous protease cleavage site. A targeting domain includes, without
limitation, a sensory neuron targeting domain, a sympathetic neuron
targeting domain, or a parasympathetic neuron targeting domain.

[0008] Other aspects of the present specification disclose uses of a TEM
disclosed herein in the manufacturing a medicament for treating a
neuroendocrine disorder disclosed herein in an individual in need
thereof.

[0009] Yet other aspects of the present specification uses of a TEM
disclosed herein in the treatment of a neuroendocrine disorder disclosed
herein in an individual in need thereof.

[0010] Other aspects of the present specification disclose methods of
treating a neuroendocrine disorder in an individual, the methods
comprising the step of administering to the individual in need thereof a
therapeutically effective amount of a composition including a Clostridial
neurotoxin and a TEM, wherein administration of the composition reduces a
symptom of the neuroendocrine, thereby treating the individual. A
Clostridial neurotoxin includes, without limitation, a Botulinum toxin
(BoNT), a Tetanus toxin (TeNT), a Baratii toxin (BaNT), and a Butyricum
toxin (BuNT). In some aspects, a TEM may comprise a targeting domain, a
Clostridial toxin translocation domain and a Clostridial toxin enzymatic
domain. In some aspects, a TEM may comprise a targeting domain, a
Clostridial toxin translocation domain, a Clostridial toxin enzymatic
domain, and an exogenous protease cleavage site. A targeting domain
includes, without limitation, a sensory neuron targeting domain, a
sympathetic neuron targeting domain, or a parasympathetic neuron
targeting domain.

[0011] Other aspects of the present specification disclose uses of a
Clostridial neurotoxin and a TEM disclosed herein in the manufacturing a
medicament for treating a neuroendocrine disorder disclosed herein in an
individual in need thereof.

[0012] Yet other aspects of the present specification uses of a
Clostridial neurotoxin and a TEM disclosed herein in the treatment of a
neuroendocrine disorder disclosed herein in an individual in need
thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows a schematic of the current paradigm of
neurotransmitter release and Clostridial toxin intoxication in a central
and peripheral neuron. FIG. 1A shows a schematic for the neurotransmitter
release mechanism of a central and peripheral neuron. The release process
can be described as comprising two steps: 1) vesicle docking, where the
vesicle-bound SNARE protein of a vesicle containing neurotransmitter
molecules associates with the membrane-bound SNARE proteins located at
the plasma membrane; and 2) neurotransmitter release, where the vesicle
fuses with the plasma membrane and the neurotransmitter molecules are
exocytosed. FIG. 1B shows a schematic of the intoxication mechanism for
tetanus and botulinum toxin activity in a central and peripheral neuron.
This intoxication process can be described as comprising four steps: 1)
receptor binding, where a Clostridial toxin binds to a Clostridial
receptor system and initiates the intoxication process; 2) complex
internalization, where after toxin binding, a vesicle containing the
toxin/receptor system complex is endocytosed into the cell; 3) light
chain translocation, where multiple events are thought to occur,
including, e.g., changes in the internal pH of the vesicle, formation of
a channel pore comprising the HN domain of the Clostridial toxin heavy
chain, separation of the Clostridial toxin light chain from the heavy
chain, and release of the active light chain and 4) enzymatic target
modification, where the activate light chain of Clostridial toxin
proteolytically cleaves its target SNARE substrate, such as, e.g.,
SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking and
neurotransmitter release.

[0014]FIG. 2 shows the domain organization of naturally-occurring
Clostridial toxins. The single-chain form depicts the amino to carboxyl
linear organization comprising an enzymatic domain, a translocation
domain, and a retargeted peptide binding domain. The di-chain loop region
located between the translocation and enzymatic domains is depicted by
the double SS bracket. This region comprises an endogenous di-chain loop
protease cleavage site that upon proteolytic cleavage with a
naturally-occurring protease, such as, e.g., an endogenous Clostridial
toxin protease or a naturally-occurring protease produced in the
environment, converts the single-chain form of the toxin into the
di-chain form. Above the single-chain form, the HCC region of the
Clostridial toxin binding domain is depicted. This region comprises the
β-trefoil domain which comprises in an amino to carboxyl linear
organization an α-fold, a β4/β5 hairpin turn, a
β-fold, a β8/β9 hairpin turn and a γ-fold.

[0015] FIG. 3 shows TEM domain organization with a targeting domain
located at the amino terminus of a TEM. FIG. 3A depicts the single-chain
polypeptide form of a TEM with an amino to carboxyl linear organization
comprising a targeting domain, a translocation domain, a di-chain loop
region comprising an exogenous protease cleavage site (P), and an
enzymatic domain. Upon proteolytic cleavage with a P protease, the
single-chain form of the TEM is converted to the di-chain form. FIG. 3B
depicts the single polypeptide form of a TEM with an amino to carboxyl
linear organization comprising a targeting domain, an enzymatic domain, a
di-chain loop region comprising an exogenous protease cleavage site (P),
and a translocation domain. Upon proteolytic cleavage with a P protease,
the single-chain form of the TEM is converted to the di-chain form.

[0016] FIG. 4 shows a TEM domain organization with a targeting domain
located between the other two domains. FIG. 4A depicts the single
polypeptide form of a TEM with an amino to carboxyl linear organization
comprising an enzymatic domain, a di-chain loop region comprising an
exogenous protease cleavage site (P), a targeting domain, and a
translocation domain. Upon proteolytic cleavage with a P protease, the
single-chain form of the TEM is converted to the di-chain form. FIG. 4B
depicts the single polypeptide form of a TEM with an amino to carboxyl
linear organization comprising a translocation domain, a di-chain loop
region comprising an exogenous protease cleavage site (P), a targeting
domain, and an enzymatic domain. Upon proteolytic cleavage with a P
protease, the single-chain form of the TEM is converted to the di-chain
form. FIG. 4c depicts the single polypeptide form of a TEM with an amino
to carboxyl linear organization comprising an enzymatic domain, a
targeting domain, a di-chain loop region comprising an exogenous protease
cleavage site (P), and a translocation domain. Upon proteolytic cleavage
with a P protease, the single-chain form of the TEM is converted to the
di-chain form. FIG. 4D depicts the single polypeptide form of a TEM with
an amino to carboxyl linear organization comprising a translocation
domain, a targeting domain, a di-chain loop region comprising an
exogenous protease cleavage site (P), and an enzymatic domain. Upon
proteolytic cleavage with a P protease, the single-chain form of the TEM
is converted to the di-chain form.

[0017] FIG. 5 shows a TEM domain organization with a targeting domain
located at the carboxyl terminus of the TEM. FIG. 5A depicts the single
polypeptide form of a TEM with an amino to carboxyl linear organization
comprising an enzymatic domain, a di-chain loop region comprising an
exogenous protease cleavage site (P), a translocation domain, and a
targeting domain. Upon proteolytic cleavage with a P protease, the
single-chain form of the TEM is converted to the di-chain form. FIG. 5B
depicts the single polypeptide form of a TEM with an amino to carboxyl
linear organization comprising a translocation domain, a di-chain loop
region comprising an exogenous protease cleavage site (P), an enzymatic
domain, and a targeting domain. Upon proteolytic cleavage with a P
protease, the single-chain form of the TEM is converted to the di-chain
form.

DESCRIPTION

[0018] Clostridia toxins produced by Clostridium botulinum, Clostridium
tetani, Clostridium baratii and Clostridium butyricum are the most widely
used in therapeutic and cosmetic treatments of humans and other mammals.
Strains of C. botulinum produce seven antigenically-distinct types of
Botulinum toxins (BoNTs), which have been identified by investigating
botulism outbreaks in man (BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals
(BoNT/C1 and BoNT/D), or isolated from soil (BoNT/G). BoNTs possess
approximately 35% amino acid identity with each other and share the same
functional domain organization and overall structural architecture. It is
recognized by those of skill in the art that within each type of
Clostridial toxin there can be subtypes that differ somewhat in their
amino acid sequence, and also in the nucleic acids encoding these
proteins. For example, there are presently five BoNT/A subtypes, BoNT/A1,
BoNT/A2, BoNT/A3 BoNT/A4 and BoNT/A5, with specific subtypes showing
approximately 89% amino acid identity when compared to another BoNT/A
subtype. While all seven BoNT serotypes have similar structure and
pharmacological properties, each also displays heterogeneous
bacteriological characteristics. In contrast, tetanus toxin (TeNT) is
produced by a uniform group of C. tetani. Two other Clostridia species,
C. baratii and C. butyricum, produce toxins, BaNT and BuNT, which are
functionally similar to BoNT/F and BoNT/E, respectively.

[0019] Clostridial toxins are released by Clostridial bacterium as
complexes comprising the approximately 150-kDa Clostridial toxin along
with associated non-toxin proteins (NAPs). Identified NAPs include
proteins possessing hemagglutination activity, such, e.g., a
hemagglutinin of approximately 17-kDa (HA-17), a hemagglutinin of
approximately 33-kDa (HA-33) and a hemagglutinin of approximately 70-kDa
(HA-70); as well as non-toxic non-hemagglutinin (NTNH), a protein of
approximately 130-kDa. Thus, the botulinum toxin type A complex can be
produced by Clostridial bacterium as 900-kDa, 500-kDa and 300-kDa forms.
Botulinum toxin types B and C1 are apparently produced as only a
500-kDa complex. Botulinum toxin type D is produced as both 300-kDa and
500-kDa complexes. Finally, botulinum toxin types E and F are produced as
only approximately 300-kDa complexes. The differences in molecular weight
for the complexes are due to differing ratios of NAPs. The toxin complex
is important for the intoxication process because it provides protection
from adverse environmental conditions, resistance to protease digestion,
and appears to facilitate internalization and activation of the toxin.

[0020] A Clostridial toxin itself is translated as a single chain
polypeptide that is subsequently cleaved by proteolytic scission within a
disulfide loop by a naturally-occurring protease (FIG. 1). This cleavage
occurs within the discrete di-chain loop region created between two
cysteine residues that form a disulfide bridge. This posttranslational
processing yields a di-chain molecule comprising an approximately 50 kDa
light chain (LC) and an approximately 100 kDa heavy chain (HC) held
together by the single disulfide bond and non-covalent interactions
between the two chains. The naturally-occurring protease used to convert
the single chain molecule into the di-chain is currently not known. In
some serotypes, such as, e.g., BoNT/A, the naturally-occurring protease
is produced endogenously by the bacteria serotype and cleavage occurs
within the cell before the toxin is release into the environment.
However, in other serotypes, such as, e.g., BoNT/E, the bacterial strain
appears not to produce an endogenous protease capable of converting the
single chain form of the toxin into the di-chain form. In these
situations, the toxin is released from the cell as a single-chain toxin
which is subsequently converted into the di-chain form by a
naturally-occurring protease found in the environment.

[0021] Each mature di-chain molecule of a Clostridial toxin comprises
three functionally distinct domains: 1) an enzymatic domain located in
the light chain (LC) that includes a metalloprotease region containing a
zinc-dependent endopeptidase activity which specifically targets core
components of the neurotransmitter release apparatus; 2) a translocation
domain contained within the amino-terminal half of the heavy chain
(HN) that facilitates release of the LC from intracellular vesicles
into the cytoplasm of the target cell; and 3) a binding domain found
within the carboxyl-terminal half of the heavy chain (HC) that
determines the binding activity and binding specificity of the toxin to
the receptor complex located at the surface of the target cell. The
HC domain comprises two distinct structural features of roughly
equal size that indicate function and are designated the HCN and
HCC subdomains.

[0022] Clostridial toxins act on the nervous system by blocking the
release of acetylcholine (ACh) at the pre-synaptic neuromuscular
junction. The binding, translocation and enzymatic activity of these
three functional domains are all necessary for toxicity. While all
details of this process are not yet precisely known, the overall cellular
intoxication mechanism whereby Clostridial toxins enter a neuron and
inhibit neurotransmitter release is similar, regardless of serotype or
subtype. Although applicants have no wish to be limited by the following
description, the intoxication mechanism can be described as comprising at
least four steps: 1) receptor binding, 2) complex internalization, 3)
light chain translocation, and 4) enzymatic target modification (FIG. 1).
The process is initiated when the binding domain of a Clostridial toxin
binds to a toxin-specific receptor system located on the plasma membrane
surface of a target cell. The binding specificity of a receptor complex
is thought to be achieved, in part, by specific combinations of
gangliosides and protein receptors that appear to distinctly comprise
each Clostridial toxin receptor complex. Once bound, the toxin/receptor
complexes are internalized by endocytosis and the internalized vesicles
are sorted to specific intracellular routes. The translocation step
appears to be triggered by the acidification of the vesicle compartment.
This process seems to initiate pH-dependent structural rearrangements
that increase hydrophobicity, create a pore in the vesicle membrane, and
promote formation of the di-chain form of the toxin. Once di-chain
formation occurs, light chain endopeptidase of the toxin is released from
the intracellular vesicle via the pore into the cytosol where it appears
to specifically target one of three known core components of the
neurotransmitter release apparatus. These core proteins,
vesicle-associated membrane protein (VAMP)/synaptobrevin,
synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are
necessary for synaptic vesicle docking and fusion at the nerve terminal
and constitute members of the soluble N-ethylmaleimide-sensitive
factor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/E
cleave SNAP-25 in the carboxyl-terminal region, releasing a nine or
twenty-six amino acid segment, respectively, and BoNT/C1 also cleaves
SNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B,
BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conserved
central portion of VAMP, and release the amino-terminal portion of VAMP
into the cytosol. BoNT/C1 cleaves syntaxin at a single site near the
cytosolic membrane surface.

[0023] Aspects of the present specification disclose, in part, in part, a
Clostridial toxin. As used herein, the term "Clostridial toxin" refers to
any toxin produced by a Clostridial toxin strain that can execute the
overall cellular mechanism whereby a Clostridial toxin intoxicates a cell
and encompasses the binding of a Clostridial toxin to a low or high
affinity Clostridial toxin receptor, the internalization of the
toxin/receptor complex, the translocation of the Clostridial toxin light
chain into the cytoplasm and the enzymatic modification of a Clostridial
toxin substrate. Non-limiting examples of Clostridial toxins include a
Botulinum toxin like BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a
BoNT/E, a BoNT/F, a BoNT/G, a Tetanus toxin (TeNT), a Baratii toxin
(BaNT), and a Butyricum toxin (BuNT). The BoNT/C2 cytotoxin and
BoNT/C3 cytotoxin, not being neurotoxins, are excluded from the term
"Clostridial toxin." A Clostridial toxin disclosed herein includes,
without limitation, naturally occurring Clostridial toxin variants, such
as, e.g., Clostridial toxin isoforms and Clostridial toxin subtypes;
non-naturally occurring Clostridial toxin variants, such as, e.g.,
conservative Clostridial toxin variants, non-conservative Clostridial
toxin variants, Clostridial toxin chimeric variants and active
Clostridial toxin fragments thereof, or any combination thereof.

[0024] A Clostridial toxin disclosed herein also includes a Clostridial
toxin complex. As used herein, the term "Clostridial toxin complex"
refers to a complex comprising a Clostridial toxin and non-toxin
associated proteins (NAPs), such as, e.g., a Botulinum toxin complex, a
Tetanus toxin complex, a Baratii toxin complex, and a Butyricum toxin
complex. Non-limiting examples of Clostridial toxin complexes include
those produced by a Clostridium botulinum, such as, e.g., a 900-kDa
BoNT/A complex, a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a
500-kDa BoNT/B complex, a 500-kDa BoNT/C1 complex, a 500-kDa BoNT/D
complex, a 300-kDa BoNT/D complex, a 300-kDa BoNT/E complex, and a
300-kDa BoNT/F complex.

[0025] Clostridial toxins can be produced using standard purification or
recombinant biology techniques known to those skilled in the art. See,
e.g., Hui Xiang et al., Animal Product Free System and Process for
Purifying a Botulinum Toxin, U.S. Pat. No. 7,354,740, which is hereby
incorporated by reference in its entirety. For example, a BoNT/A complex
can be isolated and purified from an anaerobic fermentation by
cultivating Clostridium botulinum type A in a suitable medium. Raw toxin
can be harvested by precipitation with sulfuric acid and concentrated by
ultramicrofiltration. Purification can be carried out by dissolving the
acid precipitate in calcium chloride. The toxin can then be precipitated
with cold ethanol. The precipitate can be dissolved in sodium phosphate
buffer and centrifuged. Upon drying there can then be obtained
approximately 900 kD crystalline BoNT/A complex with a specific potency
of 3×107 LD50 U/mg or greater. Furthermore, NAPs can be
separated out to obtain purified toxin, such as e.g., BoNT/A with an
approximately 150 kD molecular weight with a specific potency of
1-2×108 LD50 U/mg or greater, purified BoNT/B with an
approximately 156 kD molecular weight with a specific potency of
1-2×108 LD50 U/mg or greater, and purified BoNT/F with an
approximately 155 kD molecular weight with a specific potency of
1-2×107 LD50 U/mg or greater. See Edward J. Schantz &
Eric A. Johnson, Properties and use of Botulinum Toxin and Other
Microbial Neurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992), which
is hereby incorporated in its entirety. As another example, recombinant
Clostridial toxins can be recombinantly produced as described in Steward
et al., Optimizing Expression of Active Botulinum Toxin Type A, U.S.
Patent Publication 2008/0057575; and Steward et al., Optimizing
Expression of Active Botulinum Toxin Type E, U.S. Patent Publication
2008/0138893, each of which is hereby incorporated in its entirety.

[0027] In an embodiment, a Clostridial may be a Botulinum toxin, Tetanus
toxin, a Baratii toxin, or a Butyricum toxin. In aspects of this
embodiment, a Botulinum toxin may be a BoNT/A, a BoNT/B, a BoNT/C1,
a BoNT/D, a BoNT/E, a BoNT/F, or a BoNT/G. In another embodiment, a
Clostridial toxin may be a Clostridial toxin variant. In aspects of this
embodiment, a Clostridial toxin variant may be a naturally-occurring
Clostridial toxin variant or a non-naturally-occurring Clostridial toxin
variant. In other aspects of this embodiment, a Clostridial toxin variant
may be a BoNT/A variant, a BoNT/B variant, a BoNT/C1 variant, a
BoNT/D variant, a BoNT/E variant, a BoNT/F variant, a BoNT/G variant, a
TeNT variant, a BaNT variant, or a BuNT variant, where the variant is
either a naturally-occurring variant or a non-naturally-occurring
variant.

[0028] In an embodiment, a Clostridial toxin may be a Clostridial toxin
complex. In aspects of this embodiment, a Clostridial toxin complex may
be a BoNT/A complex, a BoNT/B complex, a BoNT/C1 complex, a BoNT/D
complex, a BoNT/E complex, a BoNT/F complex, a BoNT/G complex, a TeNT
complex, a BaNT complex, or a BuNT complex. In other aspects of this
embodiment, a Clostridial toxin complex may be a 900-kDa BoNT/A complex,
a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/B
complex, a 500-kDa BoNT/C1 complex, a 500-kDa BoNT/D complex, a 300-kDa
BoNT/D complex, a 300-kDa BoNT/E complex, or a 300-kDa BoNT/F complex.

[0029] Aspects of the present disclosure comprise, in part, a Targeted
Exocytosis Modulator. As used herein, the term "Targeted Exocytosis
Modulator" is synonymous with "TEM" or "retargeted endopeptidase."
Generally, a TEM comprises an enzymatic domain from a Clostridial toxin
light chain, a translocation domain from a Clostridial toxin heavy chain,
and a targeting domain. The targeting domain of a TEM provides an altered
cell targeting capability that targets the molecule to a receptor other
than the native Clostridial toxin receptor utilized by a
naturally-occurring Clostridial toxin. This re-targeted capability is
achieved by replacing the naturally-occurring binding domain of a
Clostridial toxin with a targeting domain having a binding activity for a
non-Clostridial toxin receptor. Although binding to a non-Clostridial
toxin receptor, a TEM undergoes all the other steps of the intoxication
process including internalization of the TEM/receptor complex into the
cytoplasm, formation of the pore in the vesicle membrane and di-chain
molecule, translocation of the enzymatic domain into the cytoplasm, and
exerting a proteolytic effect on a component of the SNARE complex of the
target cell.

[0030] However, an important difference between TEMs, such as, e.g., TEMs
disclosed herein, and native Clostridial toxins is that since TEMs do not
target motor neurons, the lethality associated with over-dosing an
individual with a TEM is greatly minimized, if not avoided altogether.
For example, a TEM comprising an opioid targeting domain can be
administered at 10,000 times the therapeutically effective dose before
evidence of lethality is observed, and this lethality is due to the
passive diffusion of the molecule and not via the intoxication process.
Thus, for all practical purposes TEMs are non-lethal molecules.

[0031] As used herein, the term "Clostridial toxin enzymatic domain"
refers to a Clostridial toxin polypeptide located in the light chain of a
Clostridial toxin that executes the enzymatic target modification step of
the intoxication process. A Clostridial toxin enzymatic domain includes a
metalloprotease region containing a zinc-dependent endopeptidase activity
which specifically targets core components of the neurotransmitter
release apparatus. Thus, a Clostridial toxin enzymatic domain
specifically targets and proteolytically cleavages of a Clostridial toxin
substrate, such as, e.g., SNARE proteins like a SNAP-25 substrate, a VAMP
substrate and a Syntaxin substrate.

[0033] As used herein, the term "Clostridial toxin translocation domain"
refers to a Clostridial toxin polypeptide located within the
amino-terminal half of the heavy chain of a Clostridial toxin that
executes the translocation step of the intoxication process. The
translocation step appears to involve an allosteric conformational change
of the translocation domain caused by a decrease in pH within the
intracellular vesicle. This conformational change results in the
formation of a pore in the vesicular membrane that permits the movement
of the light chain from within the vesicle into the cytoplasm. Thus, a
Clostridial toxin translocation domain facilitates the movement of a
Clostridial toxin light chain across a membrane of an intracellular
vesicle into the cytoplasm of a cell.

[0035] As used herein, the term "targeting domain" is synonymous with
"binding domain" or "targeting moiety" and refers to a polypeptide that
executes the receptor binding and/or complex internalization steps of the
intoxication process, with the proviso that the binding domain is not a
Clostridial toxin binding domain found within the carboxyl-terminal half
of the heavy chain of a Clostridial toxin. A targeting domain includes a
receptor binding region that confers the binding activity and/or
specificity of the targeting domain for its cognate receptor. As used
herein, the term "cognate receptor" refers to a receptor for which the
targeting domain preferentially interacts with under physiological
conditions, or under in vitro conditions substantially approximating
physiological conditions. As used herein, the term "preferentially
interacts" is synonymous with "preferentially binding" and refers to an
interaction that is statistically significantly greater in degree
relative to a control. With reference to a targeting domain disclosed
herein, a targeting domain binds to its cognate receptor to a
statistically significantly greater degree relative to a non-cognate
receptor. Said another way, there is a discriminatory binding of the
targeting domain to its cognate receptor relative to a non-cognate
receptor. Thus, a targeting domain directs binding to a TEM-specific
receptor located on the plasma membrane surface of a target cell.

[0036] In an embodiment, a targeting domain disclosed herein has an
association rate constant that confers preferential binding to its
cognate receptor. In aspects of this embodiment, a targeting domain
disclosed herein binds to its cognate receptor with an association rate
constant of, e.g., less than 1×105 M-1 s-1, less
than 1×106 M-1 s-1, less than 1×107
M-1 s-1, or less than 1×108 M-1 s-1. In
other aspects of this embodiment, a targeting domain disclosed herein
binds to its cognate receptor with an association rate constant of, e.g.,
more than 1×105 M-1 s-1, more than 1×106
M-1 s-1, more than 1×107 M-1 s-1, or more
than 1×108 M-1 s-1. In yet other aspects of this
embodiment, a targeting domain disclosed herein binds to its cognate
receptor with an association rate constant between 1×105
M-1 s-1 to 1×108 M-1 s-1, 1×106
M-1 s-1 to 1×108 M-1 s-1, 1×105
M-1 s-1 to 1×107 M-1 s-1, or
1×106 M-1 s-1 to 1×107 M-1 s-1.

[0037] In another embodiment, a targeting domain disclosed herein has an
association rate constant that is greater for its cognate target receptor
relative to a non-cognate receptor. In other aspects of this embodiment,
a targeting domain disclosed herein has an association rate constant that
is greater for its cognate target receptor relative to a non-cognate
receptor by, at least one-fold, at least two-fold, at least three-fold,
at least four fold, at least five-fold, at least 10 fold, at least 50
fold, at least 100 fold, at least 1000 fold, at least 10,000 fold, or at
least 100,000 fold. In other aspects of this embodiment, a targeting
domain disclosed herein has an association rate constant that is greater
for its cognate target receptor relative to a non-cognate receptor by,
e.g., about one-fold to about three-fold, about one-fold to about
five-fold, about one-fold to about 10-fold, about one-fold to about
100-fold, about one-fold to about 1000-fold, about five-fold to about
10-fold, about five-fold to about 100-fold, about five-fold to about
1000-fold, about 10-fold to about 100-fold, about 10-fold to about
1000-fold, about 10-fold to about 10,000-fold, or about 10-fold to about
100,000-fold.

[0038] In yet another embodiment, a targeting domain disclosed herein has
a disassociation rate constant that confers preferential binding to its
cognate receptor. In other aspects of this embodiment, a targeting domain
disclosed herein binds to its cognate receptor with a disassociation rate
constant of less than 1×10-3 s-1, less than
1×10-4 s-1, or less than 1×10-5 s-1. In
yet other aspects of this embodiment, a targeting domain disclosed herein
binds to its cognate receptor with a disassociation rate constant of,
e.g., less than 1.0×10-4 s-1, less than
2.0×10-4 s-1, less than 3.0×10-4 s-1,
less than 4.0×10-4 s-1, less than 5.0×10-4
s-1, less than 6.0×10-4 s-1, less than
7.0×10-4 s-1, less than 8.0×10-4 s-1, or
less than 9.0×10-4 s-1. In still other aspects of this
embodiment, a targeting domain disclosed herein binds to its cognate
receptor with a disassociation rate constant of, e.g., more than
1×10-3 s-1, more than 1×10-4 s-1, or more
than 1×10-5 s-1. In other aspects of this embodiment, a
targeting domain disclosed herein binds to its cognate receptor with a
disassociation rate constant of, e.g., more than 1.0×10-4
s-1, more than 2.0×10-4 s-1, more than
3.0×10-4 s-1, more than 4.0×10-4 s-1,
more than 5.0×10-4 s-1, more than 6.0×10-4
s-1, more than 7.0×10--4 s-1, more than
8.0×10-4 s-1, or more than 9.0×10-4 s-1.

[0039] In still another embodiment, a targeting domain disclosed herein
has a disassociation rate constant that is less for its cognate target
receptor relative to a non-cognate receptor. In other aspects of this
embodiment, a targeting domain disclosed herein has a disassociation rate
constant that is less for its cognate target receptor relative to a
non-cognate receptor by, e.g., at least one-fold, at least two-fold, at
least three-fold, at least four fold, at least five-fold, at least 10
fold, at least 50 fold, at least 100 fold, at least 1000 fold, at least
10,000 fold, or at least 100,000 fold. In other aspects of this
embodiment, a targeting domain disclosed herein has a disassociation rate
constant that is less for its cognate target receptor relative to a
non-cognate receptor by, e.g., about one-fold to about three-fold, about
one-fold to about five-fold, about one-fold to about 10-fold, about
one-fold to about 100-fold, about one-fold to about 1000-fold, about
five-fold to about 10-fold, about five-fold to about 100-fold, about
five-fold to about 1000-fold, about 10-fold to about 100-fold, about
10-fold to about 1000-fold, about 10-fold to about 10,000-fold, or about
10-fold to about 100,000-fold.

[0040] In another embodiment, a targeting domain disclosed herein has an
equilibrium disassociation constant that confers preferential binding to
its cognate receptor. In other aspects of this embodiment, a targeting
domain disclosed herein binds to its cognate receptor with an equilibrium
disassociation constant of, e.g., less than 0.500 nM. In yet other
aspects of this embodiment, a targeting domain disclosed herein binds to
its cognate receptor with an equilibrium disassociation constant of,
e.g., less than 0.500 nM, less than 0.450 nM, less than 0.400 nM, less
than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200
nM, less than 0.150 nM, less than 0.100 nM, or less than 0.050 nM. In
other aspects of this embodiment, a targeting domain disclosed herein
binds to its cognate receptor with an equilibrium disassociation constant
of, e.g., more than 0.500 nM, more than 0.450 nM, more than 0.400 nM,
more than 0.350 nM, more than 0.300 nM, more than 0.250 nM, more than
0.200 nM, more than 0.150 nM, more than 0.100 nM, or more than 0.050 nM.

[0041] In yet another embodiment, a targeting domain disclosed herein has
an equilibrium disassociation constant that is greater for its cognate
target receptor relative to a non-cognate receptor. In other aspects of
this embodiment, a targeting domain disclosed herein has an equilibrium
disassociation constant that is greater for its cognate target receptor
relative to a non-cognate receptor by, e.g., at least one-fold, at least
two-fold, at least three-fold, at least four fold, at least five-fold, at
least 10 fold, at least 50 fold, at least 100 fold, at least 1000 fold,
at least 10,000 fold, or at least 100,000 fold. In other aspects of this
embodiment, a targeting domain disclosed herein has an equilibrium
disassociation constant that is greater for its cognate target receptor
relative to a non-cognate receptor by, e.g., about one-fold to about
three-fold, about one-fold to about five-fold, about one-fold to about
10-fold, about one-fold to about 100-fold, about one-fold to about
1000-fold, about five-fold to about 10-fold, about five-fold to about
100-fold, about five-fold to about 1000-fold, about 10-fold to about
100-fold, about 10-fold to about 1000-fold, about 10-fold to about
10,000-fold, or about 10-fold to about 100,000-fold.

[0042] In another embodiment, a targeting domain disclosed herein may be
one that preferentially interacts with a receptor located on a sensory
neuron. In an aspect of this embodiment, the sensory neuron targeting
domain is one whose cognate receptor is located exclusively on the plasma
membrane of sensory neurons. In another aspect of this embodiment, the
sensory neuron targeting domain is one whose cognate receptor is located
primarily on the plasma membrane of sensory neuron. For example, a
receptor for a sensory neuron targeting domain is located primarily on a
sensory neuron when, e.g., at least 60% of all cells that have a cognate
receptor for a sensory neuron targeting domain on the surface of the
plasma membrane are sensory neurons, at least 70% of all cells that have
a cognate receptor for a sensory neuron targeting domain on the surface
of the plasma membrane are sensory neurons, at least 80% of all cells
that have a cognate receptor for a sensory neuron targeting domain on the
surface of the plasma membrane are sensory neurons, or at least 90% of
all cells that have a cognate receptor for a sensory neuron targeting
domain on the surface of the plasma membrane are sensory neurons. In yet
another aspect of this embodiment, the sensory neuron targeting domain is
one whose cognate receptor is located on the plasma membrane of several
types of cells, including sensory neurons. In still another aspect of
this embodiment, the sensory neuron targeting domain is one whose cognate
receptor is located on the plasma membrane of several types of cells,
including sensory neurons, with the proviso that motor neurons are not
one of the other types of cells.

[0043] In another embodiment, a targeting domain disclosed herein may be
one that preferentially interacts with a receptor located on a
sympathetic neuron. In an aspect of this embodiment, the sympathetic
neuron targeting domain is one whose cognate receptor is located
exclusively on the plasma membrane of sympathetic neurons. In another
aspect of this embodiment, the sympathetic neuron targeting domain is one
whose cognate receptor is located primarily on the plasma membrane of
sympathetic neuron. For example, a receptor for a sympathetic neuron
targeting domain is located primarily on a sympathetic neuron when, e.g.,
at least 60% of all cells that have a cognate receptor for a sympathetic
neuron targeting domain on the surface of the plasma membrane are
sympathetic neurons, at least 70% of all cells that have a cognate
receptor for a sympathetic neuron targeting domain on the surface of the
plasma membrane are sympathetic neurons, at least 80% of all cells that
have a cognate receptor for a sympathetic neuron targeting domain on the
surface of the plasma membrane are sympathetic neurons, or at least 90%
of all cells that have a cognate receptor for a sympathetic neuron
targeting domain on the surface of the plasma membrane are sympathetic
neurons. In yet another aspect of this embodiment, the sympathetic neuron
targeting domain is one whose cognate receptor is located on the plasma
membrane of several types of cells, including sympathetic neurons. In
still another aspect of this embodiment, the sympathetic neuron targeting
domain is one whose cognate receptor is located on the plasma membrane of
several types of cells, including sympathetic neurons, with the proviso
that motor neurons are not one of the other types of cells.

[0044] In another embodiment, a targeting domain disclosed herein may be
one that preferentially interacts with a receptor located on a
parasympathetic neuron. In an aspect of this embodiment, the
parasympathetic neuron targeting domain is one whose cognate receptor is
located exclusively on the plasma membrane of parasympathetic neurons. In
another aspect of this embodiment, the parasympathetic neuron targeting
domain is one whose cognate receptor is located primarily on the plasma
membrane of parasympathetic neuron. For example, a receptor for a
parasympathetic neuron targeting domain is located primarily on a
parasympathetic neuron when, e.g., at least 60% of all cells that have a
cognate receptor for a parasympathetic neuron targeting domain on the
surface of the plasma membrane are parasympathetic neurons, at least 70%
of all cells that have a cognate receptor for a parasympathetic neuron
targeting domain on the surface of the plasma membrane are
parasympathetic neurons, at least 80% of all cells that have a cognate
receptor for a parasympathetic neuron targeting domain on the surface of
the plasma membrane are parasympathetic neurons, or at least 90% of all
cells that have a cognate receptor for a parasympathetic neuron targeting
domain on the surface of the plasma membrane are parasympathetic neurons.
In yet another aspect of this embodiment, the parasympathetic neuron
targeting domain is one whose cognate receptor is located on the plasma
membrane of several types of cells, including parasympathetic neurons. In
still another aspect of this embodiment, the parasympathetic neuron
targeting domain is one whose cognate receptor is located on the plasma
membrane of several types of cells, including parasympathetic neurons,
with the proviso that motor neurons are not one of the other types of
cells.

[0046] In an aspect of this embodiment, an opioid peptide targeting domain
is an enkephalin peptide, a bovine adrenomedullary-22 (BAM22) peptide, an
endomorphin peptide, an endorphin peptide, a dynorphin peptide, a
nociceptin peptide, or a hemorphin peptide. In another aspect of this
embodiment, an enkephalin peptide targeting domain is a Leu-enkephalin
peptide, a Met-enkephalin peptide, a Met-enkephalin MRGL peptide, or a
Met-enkephalin MRF peptide. In another aspect of this embodiment, a
bovine adrenomedullary-22 peptide targeting domain is a BAM22 (1-12)
peptide, a BAM22 (6-22) peptide, a BAM22 (8-22) peptide, or a BAM22
(1-22) peptide. In another aspect of this embodiment, an endomorphin
peptide targeting domain is an endomorphin-1 peptide or an endomorphin-2
peptide. In another aspect of this embodiment, an endorphin peptide
targeting domain an endorphin-α peptide, a neoendorphin-α
peptide, an endorphin-β peptide, a neoendorphin-β peptide, or
an endorphin-γ peptide. In another aspect of this embodiment, a
dynorphin peptide targeting domain is a dynorphin A peptide, a dynorphin
B (leumorphin) peptide, or a rimorphin peptide. In another aspect of this
embodiment, a nociceptin peptide targeting domain is a nociceptin RK
peptide, a nociceptin peptide, a neuropeptide 1 peptide, a neuropeptide 2
peptide, or a neuropeptide 3 peptide. In another aspect of this
embodiment, a hemorphin peptide targeting domain is a LVVH7 peptide, a
VVH7 peptide, a VH7 peptide, a H7 peptide, a LVVH6 peptide, a LVVH5
peptide, a VVH5 peptide, a LVVH4 peptide, or a LVVH3 peptide.

[0047] In an aspect of this embodiment, a galanin peptide targeting domain
is a galanin peptide, a galanin message-associated peptide (GMAP)
peptide, a galanin like protein (GALP) peptide, or an alarin peptide.

[0048] In an aspect of this embodiment, a PAR peptide targeting domain is
a PAR1 peptide, a PAR2 peptide, a PAR3 peptide and a PAR4 peptide. In an
aspect of this embodiment, a somatostatin peptide targeting domain is a
somatostatin peptide or a cortistatin peptide. In an aspect of this
embodiment, a neurotensin peptide targeting domain a neurotensin or a
neuromedin N. In an aspect of this embodiment, a SLURP peptide targeting
domain is a SLURP-1 peptide or a SLURP-2 peptide. In an aspect of this
embodiment, an angiotensin peptide targeting domain is an angiotensin
peptide.

[0049] In an aspect of this embodiment, a tachykinin peptide targeting
domain is a Substance P peptide, a neuropeptide K peptide, a neuropeptide
gamma peptide, a neurokinin A peptide, a neurokinin B peptide, a
hemokinin peptide, or a endokinin peptide. In an aspect of this
embodiment, a Neuropeptide Y related peptide targeting domain is a
Neuropeptide Y peptide, a Peptide YY peptide, Pancreatic peptide peptide,
a Pancreatic icosapeptide peptide, a Pancreatic Hormone domain peptide, a
CXCL12 peptide, and a Sjogren syndrome antigen B peptide. In an aspect of
this embodiment, a kinin peptide targeting domain is a bradykinin
peptide, a kallidin peptide, a desArg9 bradykinin peptide, a desArg10
bradykinin peptide, a kininogen peptide, gonadotropin releasing hormone 1
peptide, chemokine peptide, an arginine vasopressin peptide.

[0050] In an aspect of this embodiment, a melanocortin peptide targeting
domain comprises a melanocyte stimulating hormone peptide, an
adrenocorticotropin peptide, a lipotropin peptide, or a melanocortin
peptide derived neuropeptide. In an aspect of this embodiment, a
melanocyte stimulating hormone peptide targeting domain comprises an
α-melanocyte stimulating hormone peptide, a β-melanocyte
stimulating hormone peptide, or a γ-melanocyte stimulating hormone
peptide. In an aspect of this embodiment, an adrenocorticotropin peptide
targeting domain comprises an adrenocorticotropin or a Corticotropin-like
intermediary peptide. In an aspect of this embodiment, a lipotropin
peptide targeting domain comprises a β-lipotropin peptide or a
γ-lipotropin peptide.

[0051] In an aspect of this embodiment, a granin peptide targeting domain
comprises a chromogranin A peptide, a chromogranin B peptide, a
chromogranin C (secretogranin II) peptide, a secretogranin IV peptide, or
a secretogranin VI peptide. In an aspect of this embodiment, a
chromogranin A peptide targeting domain comprises a β-granin
peptide, a vasostatin peptide, a chromostatin peptide, a pancreastatin
peptide, a WE-14 peptide, a catestatin peptide, a parastatin peptide, or
a GE-25 peptide. In an aspect of this embodiment, a chromogranin B
peptide targeting domain comprises a GAWK peptide, an adrenomedullary
peptide, or a secretolytin peptide. In an aspect of this embodiment, a
chromogranin C peptide targeting domain comprises a secretoneurin
peptide.

[0052] In an aspect of this embodiment, a glucagons-like hormone peptide
targeting domain is a glucagon-like peptide-1, a glucagon-like peptide-2,
a glicentin, a glicentin-related peptide (GRPP), a glucagon, or an
oxyntomodulin (OXY). In an aspect of this embodiment, a secretin peptide
targeting domain is a secretin peptide. In an aspect of this embodiment,
a pituitary adenylate cyclase activating peptide targeting domain is a
pituitary adenylate cyclase activating peptide. In an aspect of this
embodiment, a growth hormone-releasing hormone peptide targeting domain a
growth hormone-releasing hormone peptide. In an aspect of this
embodiment, a vasoactive intestinal peptide targeting domain is a
vasoactive intestinal peptide-1 peptide or a vasoactive intestinal
peptide-2 peptide. In an aspect of this embodiment, a gastric inhibitory
peptide targeting domain is a gastric inhibitory peptide. In an aspect of
this embodiment, a calcitonin peptide targeting domain is a calcitonin
peptide, an amylin peptide, a calcitonin-related peptide α, a
calcitonin-related peptide β, and a islet amyloid peptide. In an
aspect of this embodiment, a visceral gut peptide targeting domain is a
gastrin peptide, a gastrin-releasing peptide, or a cholecystokinin
peptide.

[0053] In an aspect of this embodiment, a neurotrophin peptide targeting
domain is a nerve growth factor (NGF) peptide, a brain derived
neurotrophic factor (BDNF) peptide, a neurotrophin-3 (NT-3) peptide, a
neurotrophin-4/5 (NT-4/5) peptide, or an amyloid beta (A4) precursor
protein neurotrophin (APP) peptide. In an aspect of this embodiment, a
head activator peptide targeting domain is a head activator peptide. In
an aspect of this embodiment, a glial cell line-derived neurotrophic
factor family of ligands peptide targeting domain is a glial cell
line-derived neurotrophic factor peptide, a Neurturin peptide, a
Persephrin peptide, or an Artemin peptide. In an aspect of this
embodiment, a RF-amide related peptide targeting domain a RF-amide
related peptide-1, a RF-amide related peptide-2, a RF-amide related
peptide-3, a neuropeptide AF, or a neuropeptide FF.

[0054] In an aspect of this embodiment, a neurohormone peptide targeting
domain is a corticotropin-releasing hormone (CCRH), a parathyroid hormone
(PTH), a parathyroid hormone-like hormone (PTHLH), a PHYH, a
thyrotropin-releasing hormone (TRH), an urocortin-1 (UCN1), an
urocortin-2 (UCN2), an urocortin-3 (UCN3), or an urotensin 2 (UTS2). In
an aspect of this embodiment, a neuroregulatory cytokine peptide
targeting domain is a ciliary neurotrophic factor peptide, a
glycophorin-A peptide, a leukemia inhibitory factor peptide, a
cardiotrophin-1 peptide, a cardiotrophin-like cytokine peptide, a
neuroleukin peptide, and an onostatin M peptide. In an aspect of this
embodiment, an IL peptide targeting domain is an IL-1 peptide, an IL-2
peptide, an IL-3 peptide, an IL-4 peptide, an IL-5 peptide, an IL-6
peptide, an IL-7 peptide, an IL-8 peptide, an IL-9 peptide, an IL-10
peptide, an IL-11 peptide, an IL-12 peptide, an IL-18 peptide, an IL-32
peptide, or an IL-33 peptide.

[0055] In an aspect of this embodiment, a VEGF peptide targeting domain is
a VEGF-A peptide, a VEGF-B peptide, a VEGF-C peptide, a VEGF-D peptide,
or a placenta growth factor (PIGF) peptide. In an aspect of this
embodiment, an IGF peptide targeting domain is an IGF-1 peptide or an
IGF-2 peptide. In an aspect of this embodiment, an EGF peptide targeting
domain an EGF, a heparin-binding EGF-like growth factor (HB-EGF), a
transforming growth factor-α (TGF-α), an amphiregulin (AR),
an epiregulin (EPR), an epigen (EPG), a betacellulin (BTC), a
neuregulin-1 (NRG1), a neuregulin-2 (NRG2), a neuregulin-3, (NRG3), or a
neuregulin-4 (NRG4). In an aspect of this embodiment, a FGF peptide
targeting domain is a FGF1 peptide, a FGF2 peptide, a FGF3 peptide, a
FGF4 peptide, a FGF5 peptide, a FGF6 peptide, a FGF7 peptide, a FGF8
peptide, a FGF9 peptide, a FGF10 peptide, a FGF17 peptide, or a FGF18
peptide. In an aspect of this embodiment, a PDGF peptide targeting domain
is a PDGFα peptide or a PDGFβ peptide.

[0056] In an aspect of this embodiment, a TGFβ peptide targeting
domain is a TGFβ1 peptide, a TGFβ2 peptide, a TGFβ3
peptide, or a TGFβ4 peptide. In an aspect of this embodiment, a BMP
peptide targeting domain is a BMP2 peptide, a BMP3 peptide, a BMP4
peptide, a BMP5 peptide, a BMP6 peptide, a BMP7 peptide, a BMP8 peptide,
or a BMP10 peptide. In an aspect of this embodiment, a GDF peptide
targeting domain is a GDF1 peptide, a GDF2 peptide, a GDF3 peptide, a
GDFS peptide, a GDF6 peptide, a GDF7 peptide, a GDF8 peptide, a GDF10
peptide, a GDF11 peptide, or a GDF15 peptide. In an aspect of this
embodiment, an activin peptide targeting domain is an activin A peptide,
an activin B peptide, an activin C peptide, an activin E peptide, or an
inhibin A peptide.

[0057] As discussed above, naturally-occurring Clostridial toxins are
organized into three functional domains comprising a linear
amino-to-carboxyl single polypeptide order of the enzymatic domain (amino
region position), the translocation domain (middle region position) and
the binding domain (carboxyl region position)(FIG. 2). This
naturally-occurring order can be referred to as the carboxyl presentation
of the binding domain because the domain necessary for binding to the
receptor is located at the carboxyl region position of the Clostridial
toxin. However, it has been shown that Clostridial toxins can be modified
by rearranging the linear amino-to-carboxyl single polypeptide order of
the three major domains and locating a targeting moiety at the amino
region position of a Clostridial toxin, referred to as amino
presentation, as well as in the middle region position, referred to as
central presentation (FIG. 4).

[0058] Thus, a TEM can comprise a targeting domain in any and all
locations with the proviso that TEM is capable of performing the
intoxication process. Non-limiting examples include, locating a targeting
domain at the amino terminus of a TEM; locating a targeting domain
between a Clostridial toxin enzymatic domain and a Clostridial toxin
translocation domain of a TEM; and locating a targeting domain at the
carboxyl terminus of a TEM. Other non-limiting examples include, locating
a targeting domain between a Clostridial toxin enzymatic domain and a
Clostridial toxin translocation domain of a TEM. The enzymatic domain of
naturally-occurring Clostridial toxins contains the native start
methionine. Thus, in domain organizations where the enzymatic domain is
not in the amino-terminal location an amino acid sequence comprising the
start methionine should be placed in front of the amino-terminal domain.
Likewise, where a targeting domain is in the amino-terminal position, an
amino acid sequence comprising a start methionine and a protease cleavage
site may be operably-linked in situations in which a targeting domain
requires a free amino terminus, see, e.g., Shengwen Li et al., Degradable
Clostridial Toxins, U.S. patent application Ser. No. 11/572,512 (Jan. 23,
2007), which is hereby incorporated by reference in its entirety. In
addition, it is known in the art that when adding a polypeptide that is
operably-linked to the amino terminus of another polypeptide comprising
the start methionine that the original methionine residue can be deleted.

[0059] A TEM disclosed herein may optionally comprise an exogenous
protease cleavage site that allows the use of an exogenous protease to
convert the single-chain polypeptide form of a TEM into its more active
di-chain form. As used herein, the term "exogenous protease cleavage
site" is synonymous with a "non-naturally occurring protease cleavage
site" or "non-native protease cleavage site" and means a protease
cleavage site that is not naturally found in a di-chain loop region from
a naturally occurring Clostridial toxin.

[0060] Naturally-occurring Clostridial toxins are each translated as a
single-chain polypeptide of approximately 150 kDa that is subsequently
cleaved by proteolytic scission within a disulfide loop by a
naturally-occurring protease (FIG. 2). This cleavage occurs within the
discrete di-chain loop region located between two cysteine residues that
form a disulfide bridge and comprising an endogenous protease cleavage
site. As used herein, the term "endogenous di-chain loop protease
cleavage site" is synonymous with a "naturally occurring di-chain loop
protease cleavage site" and refers to a naturally occurring protease
cleavage site found within the di-chain loop region of a naturally
occurring Clostridial toxin. This posttranslational processing yields a
di-chain molecule comprising an approximately 50 kDa light chain,
comprising the enzymatic domain, and an approximately 100 kDa heavy
chain, comprising the translocation and cell binding domains, the light
chain and heavy chain being held together by the single disulfide bond
and non-covalent interactions (FIG. 2). Recombinantly-produced
Clostridial toxins generally substitute the naturally-occurring di-chain
loop protease cleavage site with an exogenous protease cleavage site to
facilitate production of a recombinant di-chain molecule (FIGS. 3-5). See
e.g., Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat. No.
7,419,676 (Sep. 2, 2008), which is hereby incorporated by reference.

[0061] Although TEMs vary in their overall molecular weight because the
size of the targeting domain, the activation process and its reliance on
an exogenous cleavage site is essentially the same as that for
recombinantly-produced Clostridial toxins. See e.g., Steward, et al.,
Activatable Clostridial Toxins, US 2009/0081730; Steward, et al.,
Modified Clostridial Toxins with Enhanced Translocation Capabilities and
Altered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S.
patent application Ser. No. 11/776,075; Steward, et al., Modified
Clostridial Toxins with Enhanced Translocation Capabilities and Altered
Targeting Activity for Clostridial Toxin Target Cells, US 2008/0241881,
each of which is hereby incorporated by reference. In general, the
activation process that converts the single-chain polypeptide into its
di-chain form using exogenous proteases can be used to process TEMs
having a targeting domain organized in an amino presentation, central
presentation, or carboxyl presentation arrangement. This is because for
most targeting domains the amino-terminus of the moiety does not
participate in receptor binding. As such, a wide range of protease
cleavage sites can be used to produce an active di-chain form of a TEM.
However, targeting domains requiring a free amino-terminus for receptor
binding require a protease cleavage site whose scissile bond is located
at the carboxyl terminus. The use of protease cleavage site is the design
of a TEM are described in, e.g., Steward, et al., Activatable Clostridial
toxins, US 2009/0069238; Ghanshani, et al., Modified Clostridial Toxins
Comprising an Integrated Protease Cleavage Site-Binding Domain, US
2011/0189162; and Ghanshani, et al., Methods of Intracellular Conversion
of Single-Chain Proteins into their Di-chain Form, International Patent
Application Serial No. PCT/US2011/22272, each of which is incorporated by
reference in its entirety.

[0063] Thus, in an embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising a targeting domain, a
translocation domain, an exogenous protease cleavage site and an
enzymatic domain (FIG. 3A). In an aspect of this embodiment, a TEM can
comprise an amino to carboxyl single polypeptide linear order comprising
a targeting domain, a Clostridial toxin translocation domain, an
exogenous protease cleavage site and a Clostridial toxin enzymatic
domain.

[0064] In another embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising a targeting domain, an
enzymatic domain, an exogenous protease cleavage site, and a
translocation domain (FIG. 3B). In an aspect of this embodiment, a TEM
can comprise an amino to carboxyl single polypeptide linear order
comprising a targeting domain, a Clostridial toxin enzymatic domain, an
exogenous protease cleavage site, a Clostridial toxin translocation
domain.

[0065] In yet another embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising an enzymatic domain, an
exogenous protease cleavage site, a targeting domain, and a translocation
domain (FIG. 4A). In an aspect of this embodiment, a TEM can comprise an
amino to carboxyl single polypeptide linear order comprising a
Clostridial toxin enzymatic domain, an exogenous protease cleavage site,
a targeting domain, and a Clostridial toxin translocation domain.

[0066] In yet another embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising a translocation domain, an
exogenous protease cleavage site, a targeting domain, and an enzymatic
domain (FIG. 4B). In an aspect of this embodiment, a TEM can comprise an
amino to carboxyl single polypeptide linear order comprising a
Clostridial toxin translocation domain, a targeting domain, an exogenous
protease cleavage site and a Clostridial toxin enzymatic domain.

[0067] In another embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising an enzymatic domain, a
targeting domain, an exogenous protease cleavage site, and a
translocation domain (FIG. 4c). In an aspect of this embodiment, a TEM
can comprise an amino to carboxyl single polypeptide linear order
comprising a Clostridial toxin enzymatic domain, a targeting domain, an
exogenous protease cleavage site, a Clostridial toxin translocation
domain.

[0068] In yet another embodiment, a TEM can comprise an amino to carboxyl
single polypeptide linear order comprising a translocation domain, a
targeting domain, an exogenous protease cleavage site and an enzymatic
domain (FIG. 4D). In an aspect of this embodiment, a TEM can comprise an
amino to carboxyl single polypeptide linear order comprising a
Clostridial toxin translocation domain, a targeting domain, an exogenous
protease cleavage site and a Clostridial toxin enzymatic domain.

[0069] In still another embodiment, a TEM can comprise an amino to
carboxyl single polypeptide linear order comprising an enzymatic domain,
an exogenous protease cleavage site, a translocation domain, and a
targeting domain (FIG. 5A). In an aspect of this embodiment, a TEM can
comprise an amino to carboxyl single polypeptide linear order comprising
a Clostridial toxin enzymatic domain, an exogenous protease cleavage
site, a Clostridial toxin translocation domain, and a targeting domain.

[0070] In still another embodiment, a TEM can comprise an amino to
carboxyl single polypeptide linear order comprising a translocation
domain, an exogenous protease cleavage site, an enzymatic domain and a
targeting domain, (FIG. 5B). In an aspect of this embodiment, a TEM can
comprise an amino to carboxyl single polypeptide linear order comprising
a Clostridial toxin translocation domain, a targeting domain, an
exogenous protease cleavage site and a Clostridial toxin enzymatic
domain.

[0072] Aspects of the present specification disclose, in part, a
composition. In one aspect of this embodiment, a composition comprises a
TEM as disclosed herein. In another aspect of this embodiment, a
composition comprises a Clostridial toxin and a TEM as disclosed herein.
Any of the compositions disclosed herein can be useful in a method of
treating disclosed herein, with the proviso that the composition prevents
or reduces a symptom associated with condition being treated. A
Clostridial toxin and a TEM as disclosed herein may be provided as
separate compositions or as part of a single composition. It is also
understood that the two or more different Clostridial toxins and/or TEMs
can be provided as separate compositions or as part of a single
composition.

[0073] A composition disclosed herein is generally administered as a
pharmaceutical acceptable composition. As used herein, the term
"pharmaceutically acceptable" means any molecular entity or composition
that does not produce an adverse, allergic or other untoward or unwanted
reaction when administered to an individual. As used herein, the term
"pharmaceutically acceptable composition" is synonymous with
"pharmaceutical composition" and means a therapeutically effective
concentration of an active ingredient, such as, e.g., any of the
Clostridial toxins and/or TEMs disclosed herein. A pharmaceutical
composition disclosed herein is useful for medical and veterinary
applications. A pharmaceutical composition may be administered to an
individual alone, or in combination with other supplementary active
ingredients, agents, drugs or hormones. The pharmaceutical compositions
may be manufactured using any of a variety of processes, including,
without limitation, conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, and
lyophilizing. The pharmaceutical composition can take any of a variety of
forms including, without limitation, a sterile solution, suspension,
emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup,
elixir or any other dosage form suitable for administration.

[0074] A pharmaceutical composition disclosed herein may optionally
include a pharmaceutically acceptable carrier that facilitates processing
of an active ingredient into pharmaceutically acceptable compositions. As
used herein, the term "pharmacologically acceptable carrier" is
synonymous with "pharmacological carrier" and means any carrier that has
substantially no long term or permanent detrimental effect when
administered and encompasses terms such as "pharmacologically acceptable
vehicle, stabilizer, diluent, additive, auxiliary or excipient." Such a
carrier generally is mixed with an active ingredient, or permitted to
dilute or enclose the active compound and can be a solid, semi-solid, or
liquid agent. It is understood that the active ingredients can be soluble
or can be delivered as a suspension in the desired carrier or diluent.
Any of a variety of pharmaceutically acceptable carriers can be used
including, without limitation, aqueous media such as, e.g., water,
saline, glycine, hyaluronic acid and the like; solid carriers such as,
e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like;
solvents; dispersion media; coatings; antibacterial and antifungal
agents; isotonic and absorption delaying agents; or any other inactive
ingredient. Selection of a pharmacologically acceptable carrier can
depend on the mode of administration. Except insofar as any
pharmacologically acceptable carrier is incompatible with the active
ingredient, its use in pharmaceutically acceptable compositions is
contemplated. Non-limiting examples of specific uses of such
pharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS AND
DRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams
& Wilkins Publishers, 7th ed. 1999); REMINGTON: THE SCIENCE AND
PRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams &
Wilkins, 20th ed. 2000); GOODMAN & GILMAN'S THE PHARMACOLOGICAL
BASIS OF THERAPEUTICS (Joel G. Hardman et al., eds., McGraw-Hill
Professional, 10th ed. 2001); and HANDBOOK OF PHARMACEUTICAL
EXCIPIENTS (Raymond C. Rowe et al., APhA Publications, 4th edition
2003). These protocols are routine procedures and any modifications are
well within the scope of one skilled in the art and from the teaching
herein.

[0075] A pharmaceutical composition disclosed herein can optionally
include, without limitation, other pharmaceutically acceptable components
(or pharmaceutical components), including, without limitation, buffers,
preservatives, tonicity adjusters, salts, antioxidants, osmolality
adjusting agents, physiological substances, pharmacological substances,
bulking agents, emulsifying agents, wetting agents, sweetening or
flavoring agents, and the like. Various buffers and means for adjusting
pH can be used to prepare a pharmaceutical composition disclosed herein,
provided that the resulting preparation is pharmaceutically acceptable.
Such buffers include, without limitation, acetate buffers, citrate
buffers, phosphate buffers, neutral buffered saline, phosphate buffered
saline and borate buffers. It is understood that acids or bases can be
used to adjust the pH of a composition as needed. Pharmaceutically
acceptable antioxidants include, without limitation, sodium
metabisulfite, sodium thiosulfate, acetylcysteine, butylated
hydroxyanisole and butylated hydroxytoluene. Useful preservatives
include, without limitation, benzalkonium chloride, chlorobutanol,
thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilized
oxy chloro composition and chelants, such as, e.g., DTPA or
DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide. Tonicity adjustors
useful in a pharmaceutical composition include, without limitation, salts
such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin
and other pharmaceutically acceptable tonicity adjustor. The
pharmaceutical composition may be provided as a salt and can be formed
with many acids, including but not limited to, hydrochloric, sulfuric,
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more
soluble in aqueous or other protonic solvents than are the corresponding
free base forms. It is understood that these and other substances known
in the art of pharmacology can be included in a pharmaceutical
composition. Exemplary pharmaceutical composition comprising a TEM are
described in Hunt, et al., Animal Protein-Free Pharmaceutical
Compositions, U.S. Ser. No. 12/331,816; and Dasari, et al., Clostridial
Toxin Pharmaceutical Compositions, WO/2010/090677, each of which is
hereby incorporated by reference in its entirety.

[0076] In an embodiment, a composition is a pharmaceutical composition
comprising a TEM. In aspects of this embodiment, a pharmaceutical
composition comprising a TEM further comprises a pharmacological carrier,
a pharmaceutical component, or both a pharmacological carrier and a
pharmaceutical component. In other aspects of this embodiment, a
pharmaceutical composition comprising a TEM further comprises at least
one pharmacological carrier, at least one pharmaceutical component, or at
least one pharmacological carrier and at least one pharmaceutical
component.

[0077] In another embodiment, a composition is a pharmaceutical
composition comprising a Clostridial toxin. In aspects of this
embodiment, a pharmaceutical composition comprising a Clostridial toxin
further comprises a pharmacological carrier, a pharmaceutical component,
or both a pharmacological carrier and a pharmaceutical component. In
other aspects of this embodiment, a pharmaceutical composition comprising
a Clostridial toxin further comprises at least one pharmacological
carrier, at least one pharmaceutical component, or at least one
pharmacological carrier and at least one pharmaceutical component.

[0078] In yet another embodiment, a composition is a pharmaceutical
composition comprising a Clostridial toxin and a TEM. In aspects of this
embodiment, a pharmaceutical composition comprising a Clostridial toxin
and a TEM further comprises a pharmacological carrier, a pharmaceutical
component, or both a pharmacological carrier and a pharmaceutical
component. In other aspects of this embodiment, a pharmaceutical
composition comprising a Clostridial toxin and a TEM further comprises at
least one pharmacological carrier, at least one pharmaceutical component,
or at least one pharmacological carrier and at least one pharmaceutical
component.

[0079] Aspects of the present specification disclose, in part, treating an
individual suffering from a neuroendocrine disorder. As used herein, the
term "treating," refers to reducing or eliminating in an individual a
clinical symptom of a neuroendocrine disorder; or delaying or preventing
in an individual the onset of a clinical symptom of a neuroendocrine
disorder. For example, the term "treating" can mean reducing a symptom of
a condition characterized by a neuroendocrine disorder by, e.g., at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90% or at least 100%. The actual symptoms
associated with a neuroendocrine disorder are well known and can be
determined by a person of ordinary skill in the art by taking into
account factors, including, without limitation, the location of the
neuroendocrine disorder, the cause of the neuroendocrine disorder, the
severity of the neuroendocrine disorder, and/or the tissue or organ
affected by the neuroendocrine disorder. Those of skill in the art will
know the appropriate symptoms or indicators associated with specific
neuroendocrine disorder and will know how to determine if an individual
is a candidate for treatment as disclosed herein.

[0080] As used herein, the term "neuroendocrine disorder" refers to a
neuroendocrine disorder where at least one of the underlying symptoms
being treated is due to a sensory nerve-based etiology, a sympathetic
nerve-based etiology, and/or a parasympathetic nerve-based etiology.
Typically such etiologies will involve an abnormal overactivity of a
nerve that results in symptoms of a neuroendocrine disorder, or any
normal activity of a nerve that needs to be reduced or stopped for a
period of time in order to treat a neuroendocrine disorder.

[0081] A composition or compound is administered to an individual. An
individual comprises all mammals including a human being. Typically, any
individual who is a candidate for a conventional neuroendocrine disorder
treatment is a candidate for a neuroendocrine disorder treatment
disclosed herein. Pre-operative evaluation typically includes routine
history and physical examination in addition to thorough informed consent
disclosing all relevant risks and benefits of the procedure.

[0082] With reference to a therapy comprising a TEM, the amount of a TEM
disclosed herein used with the methods of treatment disclosed herein will
typically be an effective amount. As used herein, the term "effective
amount" is synonymous with "therapeutically effective amount", "effective
dose", or "therapeutically effective dose" and when used in reference to
treating a neuroendocrine disorder means the minimum dose of a TEM alone
necessary to achieve the desired therapeutic effect and includes a dose
sufficient to reduce a symptom associated with a neuroendocrine disorder.
An effective amount refers to the total amount of a TEM administered to
an individual in one setting. As such, an effective amount of a TEM does
not refer to the amount administered per site. The effectiveness of a TEM
disclosed herein in treating a neuroendocrine disorder can be determined
by observing an improvement in an individual based upon one or more
clinical symptoms, and/or physiological indicators associated with the
condition. An improvement in a neuroendocrine disorder also can be
indicated by a reduced need for a concurrent therapy.

[0083] With reference to a standard dose combination therapy comprising a
Clostridial toxin and a TEM, an effective amount of a Clostridial toxin
is one where in combination with a TEM the amount of a Clostridial toxin
achieves the desired therapeutic effect. For example, typically about
75-150 U of BOTOX® (Allergan, Inc., Irvine, Calif.), a BoNT/A, is
administered in order to treat a neuroendocrine disorder.

[0084] With reference to a low dose combination therapy comprising a
Clostridial toxin and a TEM, an effective amount of a Clostridial toxin
is one where in combination with a TEM the amount of a Clostridial toxin
achieves the desired therapeutic effect, but such an amount administered
on its own would be ineffective. For example, typically about 75-150 U of
BOTOX® (Allergan, Inc., Irvine, Calif.), a BoNT/A, is administered in
order to treat a neuroendocrine disorder. However, in a low dose
combination therapy, a suboptimal effective amount of BoNT/A would be
administered to treat a neuroendocrine disorder when such toxin is used
in a combined therapy with a TEM. For example, less that 50 U, less than
25 U, less than 15 U, less than 10 U, less than 7.5 U, less than 5 U,
less than 2.5 U, or less than 1 U of BoNT/A would be administered to
treat a neuroendocrine disorder when used in a low dose combination
therapy with a TEM as disclosed herein.

[0085] The appropriate effective amount of a Clostridial toxin and/or a
TEM to be administered to an individual for a particular neuroendocrine
disorder can be determined by a person of ordinary skill in the art by
taking into account factors, including, without limitation, the type of
neuroendocrine disorder, the location of the neuroendocrine disorder, the
cause of the neuroendocrine disorder, the severity of the neuroendocrine
disorder, the degree of relief desired, the duration of relief desired,
the particular Clostridial toxin and/or a TEM used, the rate of excretion
of the Clostridial toxin and/or a TEM used, the pharmacodynamics of the
Clostridial toxin and/or a TEM used, the nature of the other compounds to
be included in the composition, the particular route of administration,
the particular characteristics, history and risk factors of the
individual, such as, e.g., age, weight, general health and the like, or
any combination thereof. Additionally, where repeated administration of a
composition comprising disclosed herein is used, an effective amount of a
Clostridial toxin and/or a TEM will further depend upon factors,
including, without limitation, the frequency of administration, the
half-life of the composition comprising a Clostridial toxin and/or a TEM,
or any combination thereof. In is known by a person of ordinary skill in
the art that an effective amount of a composition comprising a
Clostridial toxin and/or a TEM can be extrapolated from in vitro assays
and in vivo administration studies using animal models prior to
administration to humans.

[0086] Wide variations in the necessary effective amount are to be
expected in view of the differing efficiencies of the various routes of
administration. For instance, oral administration generally would be
expected to require higher dosage levels than administration by
intravenous or intravitreal injection. Similarly, systemic administration
of a TEM would be expected to require higher dosage levels than a local
administration. Variations in these dosage levels can be adjusted using
standard empirical routines of optimization, which are well-known to a
person of ordinary skill in the art. The precise therapeutically
effective dosage levels and patterns are preferably determined by the
attending physician in consideration of the above-identified factors. One
skilled in the art will recognize that the condition of the individual
can be monitored throughout the course of therapy and that the effective
amount of a TEM disclosed herein that is administered can be adjusted
accordingly.

[0087] In aspects of this embodiment, a therapeutically effective amount
of a composition comprising a TEM reduces a symptom associated with a
neuroendocrine disorder by, e.g., at least 10%, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90% or at least 100%. In other aspects of this embodiment,
a therapeutically effective amount of a composition comprising a TEM
reduces a symptom associated with a neuroendocrine disorder by, e.g., at
most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most
60%, at most 70%, at most 80%, at most 90% or at most 100%. In yet other
aspects of this embodiment, a therapeutically effective amount of a
composition comprising a TEM reduces a symptom associated with a
neuroendocrine disorder by, e.g., about 10% to about 100%, about 10% to
about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to
about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to
about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to
about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to
about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to
about 80%, about 30% to about 70%, about 30% to about 60%, or about 30%
to about 50%. In still other aspects of this embodiment, a
therapeutically effective amount of the TEM is the dosage sufficient to
inhibit neuronal activity for, e.g., at least one week, at least one
month, at least two months, at least three months, at least four months,
at least five months, at least six months, at least seven months, at
least eight months, at least nine months, at least ten months, at least
eleven months, or at least twelve months.

[0088] In other aspects of this embodiment, a therapeutically effective
amount of a TEM generally is in the range of about 1 fg to about 3.0 mg.
In aspects of this embodiment, an effective amount of a TEM can be, e.g.,
about 100 fg to about 3.0 mg, about 100 μg to about 3.0 mg, about 100
ng to about 3.0 mg, or about 100 μg to about 3.0 mg. In other aspects
of this embodiment, an effective amount of a TEM can be, e.g., about 100
fg to about 750 μg, about 100 μg to about 750 μg, about 100 ng
to about 750 μg, or about 1 μg to about 750 μg. In yet other
aspects of this embodiment, a therapeutically effective amount of a TEM
can be, e.g., at least 1 fg, at least 250 fg, at least 500 fg, at least
750 fg, at least 1 μg, at least 250 μg, at least 500 μg, at
least 750 μg, at least 1 ng, at least 250 ng, at least 500 ng, at
least 750 ng, at least 1 μg, at least 250 μg, at least 500 μg,
at least 750 μg, or at least 1 mg. In still other aspects of this
embodiment, a therapeutically effective amount of a composition
comprising a TEM can be, e.g., at most 1 fg, at most 250 fg, at most 500
fg, at most 750 fg, at most 1 μg, at most 250 μg, at most 500
μg, at most 750 μg, at most 1 ng, at most 250 ng, at most 500 ng,
at most 750 ng, at most 1 μg, at least 250 μg, at most 500 μg,
at most 750 μg, or at most 1 mg.

[0089] In yet other aspects of this embodiment, a therapeutically
effective amount of a TEM generally is in the range of about 0.00001
mg/kg to about 3.0 mg/kg. In aspects of this embodiment, an effective
amount of a TEM can be, e.g., about 0.0001 mg/kg to about 0.001 mg/kg,
about 0.03 mg/kg to about 3.0 mg/kg, about 0.1 mg/kg to about 3.0 mg/kg,
or about 0.3 mg/kg to about 3.0 mg/kg. In yet other aspects of this
embodiment, a therapeutically effective amount of a TEM can be, e.g., at
least 0.00001 mg/kg, at least 0.0001 mg/kg, at least 0.001 mg/kg, at
least 0.01 mg/kg, at least 0.1 mg/kg, or at least 1 mg/kg. In yet other
aspects of this embodiment, a therapeutically effective amount of a TEM
can be, e.g., at most 0.00001 mg/kg, at most 0.0001 mg/kg, at most 0.001
mg/kg, at most 0.01 mg/kg, at most 0.1 mg/kg, or at most 1 mg/kg.

[0090] In aspects of this embodiment, a therapeutically effective amount
of a composition comprising a Clostridial toxin reduces a symptom
associated with a neuroendocrine disorder by, e.g., at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90% or at least 100%. In other aspects
of this embodiment, a therapeutically effective amount of a composition
comprising a Clostridial toxin reduces a symptom associated with a
neuroendocrine disorder by, e.g., at most 10%, at most 20%, at most 30%,
at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most
90% or at most 100%. In yet other aspects of this embodiment, a
therapeutically effective amount of a composition comprising a
Clostridial toxin reduces a symptom associated with a neuroendocrine
disorder by, e.g., about 10% to about 100%, about 10% to about 90%, about
10% to about 80%, about 10% to about 70%, about 10% to about 60%, about
10% to about 50%, about 10% to about 40%, about 20% to about 100%, about
20% to about 90%, about 20% to about 80%, about 20% to about 20%, about
20% to about 60%, about 20% to about 50%, about 20% to about 40%, about
30% to about 100%, about 30% to about 90%, about 30% to about 80%, about
30% to about 70%, about 30% to about 60%, or about 30% to about 50%. In
still other aspects of this embodiment, a therapeutically effective
amount of a Clostridial toxin is the dosage sufficient to inhibit
neuronal activity for, e.g., at least one week, at least one month, at
least two months, at least three months, at least four months, at least
five months, at least six months, at least seven months, at least eight
months, at least nine months, at least ten months, at least eleven
months, or at least twelve months.

[0091] In other aspects of this embodiment, a therapeutically effective
amount of a Clostridial toxin generally is in the range of about 1 fg to
about 30.0 μg. In other aspects of this embodiment, a therapeutically
effective amount of a Clostridial toxin can be, e.g., at least 1.0 μg,
at least 10 μg, at least 100 μg, at least 1.0 ng, at least 10 ng,
at least 100 ng, at least 1.0 μg, at least 10 μg, at least 100
μg, or at least 1.0 mg. In still other aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be, e.g., at
most 1.0 μg, at most 10 μg, at most 100 μg, at most 1.0 ng, at
most 10 ng, at most 100 ng, at most 1.0 μg, at most 10 μg, at most
100 μg, or at most 1.0 mg. In still other aspects of this embodiment,
a therapeutically effective amount of a Clostridial toxin can be, e.g.,
about 1.0 μg to about 10 μg, about 10 μg to about 10 μg,
about 100 μg to about 10 μg, about 1.0 ng to about 10 μg, about
10 ng to about 10 μg, or about 100 ng to about 10 μg. In still
other aspects of this embodiment, a therapeutically effective amount of a
Clostridial toxin can be from, e.g., about 1.0 μg to about 1.0 μg,
about 10 μg to about 1.0 μg, about 100 μg to about 1.0 μg,
about 1.0 ng to about 1.0 μg, about 10 ng to about 1.0 μg, or about
100 ng to about 1.0 μg. In other aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be from,
e.g., about 1.0 μg to about 100 ng, about 10 μg to about 100 ng,
about 100 μg to about 100 ng, about 1.0 ng to about 100 ng, or about
10 ng to about 100 ng.

[0092] In yet other aspects of this embodiment, a therapeutically
effective amount of a Clostridial toxin generally is in the range of
about 0.1 U to about 2500 U. In other aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be, e.g., at
least 1.0 U, at least 10 U, at least 100 U, at least 250 U, at least 500
U, at least 750 U, at least 1,000 U, at least 1,500 U, at least 2,000 U,
or at least 2,500 U. In still other aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be, e.g., at
most 1.0 U, at most 10 U, at most 100 U, at most 250 U, at most 500 U, at
most 750 U, at most 1,000 U, at most 1,500 U, at most 2,000 U, or at most
2,500 U. In still other aspects of this embodiment, a therapeutically
effective amount of a Clostridial toxin can be, e.g., about 1 U to about
2,000 U, about 10 U to about 2,000 U, about 50 U to about 2,000 U, about
100 U to about 2,000 U, about 500 U to about 2,000 U, about 1,000 U to
about 2,000 U, about 1 U to about 1,000 U, about 10 U to about 1,000 U,
about 50 U to about 1,000 U, about 100 U to about 1,000 U, about 500 U to
about 1,000 U, about 1 U to about 500 U, about 10 U to about 500 U, about
50 U to about 500 U, about 100 U to about 500 U, about 1 U to about 100
U, about 10 U to about 100 U, about 50 U to about 100 U, about 0.1 U to
about 1 U, about 0.1 U to about 5 U, about 0.1 U to about 10 U, about 0.1
U to about 15 U, about 0.1 U to about 20 U, about 0.1 U to about 25 U.

[0093] In still other aspects of this embodiment, a therapeutically
effective amount of a Clostridial toxin generally is in the range of
about 0.0001 U/kg to about 3,000 U/kg. In aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be, e.g., at
least 0.001 U/kg, at least 0.01 U/kg, at least 0.1 U/kg, at least 1.0
U/kg, at least 10 U/kg, at least 100 U/kg, or at least 1000 U/kg. In
other aspects of this embodiment, a therapeutically effective amount of a
Clostridial toxin can be, e.g., at most 0.001 U/kg, at most 0.01 U/kg, at
most 0.1 U/kg, at most 1.0 U/kg, at most 10 U/kg, at most 100 U/kg, or at
most 1000 U/kg. In yet other aspects of this embodiment, a
therapeutically effective amount of a Clostridial toxin can be between,
e.g., about 0.001 U/kg to about 1 U/kg, about 0.01 U/kg to about 1 U/kg,
about 0.1 U/kg to about 1 U/kg, about 0.001 U/kg to about 10 U/kg, about
0.01 U/kg to about 10 U/kg, about 0.1 U/kg to about 10 U/kg about 1 U/kg
to about 10 U/kg, about 0.001 U/kg to about 100 U/kg, about 0.01 U/kg to
about 100 U/kg, about 0.1 U/kg to about 100 U/kg, about 1 U/kg to about
100 U/kg, or about 10 U/kg to about 100 U/kg. As used herein, the term
"unit" or "U" is refers to the LD50 dose, which is defined as the
amount of a Clostridial toxin disclosed herein that killed 50% of the
mice injected with the Clostridial toxin.

[0094] In aspects of this embodiment, a therapeutically effective amount
of a standard or low combination therapy comprising a Clostridial toxin
and a TEM reduces a symptom associated with a neuroendocrine disorder by,
e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90% or at least
100%. In other aspects of this embodiment, a therapeutically effective
amount of a standard or low combination therapy comprising a Clostridial
toxin and a TEM reduces a symptom associated with a neuroendocrine
disorder by, e.g., at most 10%, at most 20%, at most 30%, at most 40%, at
most 50%, at most 60%, at most 70%, at most 80%, at most 90% or at most
100%. In yet other aspects of this embodiment, a therapeutically
effective amount of a standard or low combination therapy comprising a
Clostridial toxin and a TEM reduces a symptom associated with a
neuroendocrine disorder by, e.g., about 10% to about 100%, about 10% to
about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to
about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to
about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to
about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to
about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to
about 80%, about 30% to about 70%, about 30% to about 60%, or about 30%
to about 50%. In still other aspects of this embodiment, a
therapeutically effective amount of a standard or low combination therapy
comprising a Clostridial toxin and a TEM is the dosage sufficient to
inhibit neuronal activity for, e.g., at least one week, at least one
month, at least two months, at least three months, at least four months,
at least five months, at least six months, at least seven months, at
least eight months, at least nine months, at least ten months, at least
eleven months, or at least twelve months.

[0095] In other aspects of this embodiment, a therapeutically effective
amount of a standard or low combination therapy comprising a Clostridial
toxin and a TEM generally is in a Clostridial toxin:TEM molar ratio of
about 1:1 to about 1:10,000. In other aspects of this embodiment, a
therapeutically effective amount of a standard or low combination therapy
comprising a Clostridial toxin and a TEM can be in a Clostridial
toxin:TEM molar ratio of, e.g., about 1:1, about 1:2, about 1:5, about
1:10, about 1:25, about 1:50, about 1:75, about 1:100, about 1:200, about
1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800,
about 1:900, about 1:1000, about 1:2000, about 1:3000, about 1:4000,
about 1:5000, about 1:6000, about 1:7000, about 1:8000, about 1:9000, or
about 1:10,000. In yet other aspects of this embodiment, a
therapeutically effective amount of standard or low combination therapy
comprising a Clostridial toxin and a TEM can be in a Clostridial
toxin:TEM molar ratio of, e.g., at least 1:1, at least 1:2, at least 1:5,
at least 1:10, at least 1:25, at least 1:50, at least 1:75, at least
1:100, at least 1:200, at least 1:300, at least 1:400, at least 1:500, at
least 1:600, at least 1:700, at least 1:800, at least 1:900, at least
1:1000, at least 1:2000, at least 1:3000, at least 1:4000, at least
1:5000, at least 1:6000, at least 1:7000, at least 1:8000, at least
1:9000, or at least 1:10,000. In still other aspects of this embodiment,
a therapeutically effective amount of a standard or low combination
therapy comprising a Clostridial toxin and a TEM can be in a Clostridial
toxin:TEM molar ratio of between, e.g., about 1:1 to about 1:10,000,
about 1:10 to about 1:10,000, about 1:100 to about 1:10,000, about 1:500
to about 1:10,000, about 1:1000 to about 1:10,000, about 1:5000 to about
1:10,000, about 1:1 to about 1:1000, about 1:10 to about 1:1000, about
1:100 to about 1:1000, about 1:250 to about 1:1000, about 1:500 to about
1:1000, about 1:750 to about 1:1000, about 1:1 to about 1:500, about 1:10
to about 1:500, about 1:50 to about 1:500, about 1:100 to about 1:500,
about 1:250 to about 1:500, about 1:1 to about 1:100, about 1:10 to about
1:100, about 1:25 to about 1:100, about 1:50 to about 1:100, or about
1:75 to about 1:100.

[0096] In yet other aspects of this embodiment, a therapeutically
effective amount of a standard combination therapy comprising a
Clostridial toxin and a TEM generally is in a range of about 0.50 U to
about 250 U of Clostridial toxin and about 0.1 μg to about 2,000.0
μg of a TEM. In aspects of this embodiment, a therapeutically
effective amount of a combined therapy comprising a Clostridial toxin and
a TEM can be, e.g., about 0.1 U to about 10 U of a Clostridial toxin and
about 10 μg to about 1,000 μg of a TEM, about 0.1 U to about 10 U
of a Clostridial toxin and about 10 μg to about 500 μg of a TEM,
about 0.1 U to about 10 U of a Clostridial toxin and about 10 μg to
about 100 μg of a TEM, about 0.5 U to about 10 U of a Clostridial
toxin and about 10 μg to about 1,000 μg of a TEM, about 0.5 U to
about 10 U of a Clostridial toxin and about 10 μg to about 500 μg
of a TEM, about 0.5 U to about 10 U of a Clostridial toxin and about 10
μg to about 100 μg of a TEM, about 1 U to about 10 U of a
Clostridial toxin and about 100 μg to about 1,000 μg of a TEM,
about 1 U to about 10 U of a Clostridial toxin and about 100 μg to
about 500 μg of a TEM, or about 1 U to about 10 U of a Clostridial
toxin and about 100 μg to about 100 μg of a TEM.

[0097] In yet other aspects of this embodiment, a therapeutically
effective amount of a low combination therapy comprising a Clostridial
toxin and a TEM generally is in a range of about 0.01 U to about 50 U of
Clostridial toxin and about 0.1 μg to about 2,000.0 μg of a TEM. In
aspects of this embodiment, a therapeutically effective amount of a
combined therapy comprising a Clostridial toxin and a TEM can be, e.g.,
about 0.1 U to about 10 U of a Clostridial toxin and about 10 μg to
about 1,000 μg of a TEM, about 0.1 U to about 10 U of a Clostridial
toxin and about 10 μg to about 500 μg of a TEM, about 0.1 U to
about 10 U of a Clostridial toxin and about 10 μg to about 100 μg
of a TEM, about 0.5 U to about 10 U of a Clostridial toxin and about 10
μg to about 1,000 μg of a TEM, about 0.5 U to about 10 U of a
Clostridial toxin and about 10 μg to about 500 μg of a TEM, about
0.5 U to about 10 U of a Clostridial toxin and about 10 μg to about
100 μg of a TEM, about 1 U to about 10 U of a Clostridial toxin and
about 100 μg to about 1,000 μg of a TEM, about 1 U to about 10 U of
a Clostridial toxin and about 100 μg to about 500 μg of a TEM, or
about 1 U to about 10 U of a Clostridial toxin and about 100 μg to
about 100 μg of a TEM.

[0098] Dosing can be single dosage or cumulative (serial dosing), and can
be readily determined by one skilled in the art. For instance, treatment
of a neuroendocrine disorder may comprise a one-time administration of an
effective dose of a composition disclosed herein. As a non-limiting
example, an effective dose of a composition disclosed herein can be
administered once to an individual, e.g., as a single injection or
deposition at or near the site exhibiting a symptom of a neuroendocrine
disorder. Alternatively, treatment of a neuroendocrine disorder may
comprise multiple administrations of an effective dose of a composition
disclosed herein carried out over a range of time periods, such as, e.g.,
daily, once every few days, weekly, monthly or yearly. As a non-limiting
example, a composition disclosed herein can be administered once or twice
yearly to an individual. The timing of administration can vary from
individual to individual, depending upon such factors as the severity of
an individual's symptoms. For example, an effective dose of a composition
disclosed herein can be administered to an individual once a month for an
indefinite period of time, or until the individual no longer requires
therapy. A person of ordinary skill in the art will recognize that the
condition of the individual can be monitored throughout the course of
treatment and that the effective amount of a composition disclosed herein
that is administered can be adjusted accordingly.

[0099] A composition disclosed herein can be administered to an individual
using a variety of routes. Routes of administration suitable for a method
of treating a neuroendocrine disorder as disclosed herein include both
local and systemic administration. Local administration results in
significantly more delivery of a composition to a specific location as
compared to the entire body of the individual, whereas, systemic
administration results in delivery of a composition to essentially the
entire body of the individual. Routes of administration suitable for a
method of treating a neuroendocrine disorder as disclosed herein also
include both central and peripheral administration. Central
administration results in delivery of a composition to essentially the
central nervous system of an individual and includes, e.g., intrathecal
administration, epidural administration as well as a cranial injection or
implant. Peripheral administration results in delivery of a composition
to essentially any area of an individual outside of the central nervous
system and encompasses any route of administration other than direct
administration to the spine or brain. The actual route of administration
of a composition disclosed herein used can be determined by a person of
ordinary skill in the art by taking into account factors, including,
without limitation, the type of neuroendocrine disorder, the location of
the neuroendocrine disorder, the cause of the neuroendocrine disorder,
the severity of the neuroendocrine disorder, the degree of relief
desired, the duration of relief desired, the particular Clostridial toxin
and/or TEM used, the rate of excretion of the Clostridial toxin and/or
TEM used, the pharmacodynamics of the Clostridial toxin and/or TEM used,
the nature of the other compounds to be included in the composition, the
particular route of administration, the particular characteristics,
history and risk factors of the individual, such as, e.g., age, weight,
general health and the like, or any combination thereof.

[0100] In an embodiment, a composition disclosed herein is administered
systemically to an individual. In another embodiment, a composition
disclosed herein is administered locally to an individual. In an aspect
of this embodiment, a composition disclosed herein is administered to a
nerve of an individual. In another aspect of this embodiment, a
composition disclosed herein is administered to the area surrounding a
nerve of an individual.

[0101] A composition disclosed herein can be administered to an individual
using a variety of delivery mechanisms. The actual delivery mechanism
used to administer a composition disclosed herein to an individual can be
determined by a person of ordinary skill in the art by taking into
account factors, including, without limitation, the type of
neuroendocrine disorder, the location of the neuroendocrine disorder, the
cause of the neuroendocrine disorder, the severity of the neuroendocrine
disorder, the degree of relief desired, the duration of relief desired,
the particular Clostridial toxin and/or TEM used, the rate of excretion
of the Clostridial toxin and/or TEM used, the pharmacodynamics of the
Clostridial toxin and/or TEM used, the nature of the other compounds to
be included in the composition, the particular route of administration,
the particular characteristics, history and risk factors of the
individual, such as, e.g., age, weight, general health and the like, or
any combination thereof.

[0102] In an embodiment, a composition disclosed herein is administered by
injection. In aspects of this embodiment, administration of a composition
disclosed herein is by, e.g., intramuscular injection, intraorgan
injection, subdermal injection, dermal injection, intracranical
injection, spinal injection, or injection into any other body area for
the effective administration of a composition disclosed herein. In
aspects of this embodiment, injection of a composition disclosed herein
is to a nerve or into the area surrounding a nerve.

[0103] In another embodiment, a composition disclosed herein is
administered by catheter. In aspects of this embodiment, administration
of a composition disclosed herein is by, e.g., a catheter placed in an
epidural space.

[0104] A composition disclosed herein as disclosed herein can also be
administered to an individual in combination with other therapeutic
compounds to increase the overall therapeutic effect of the treatment.
The use of multiple compounds to treat an indication can increase the
beneficial effects while reducing the presence of side effects.

[0105] Aspects of the present invention can also be described as follows:
[0106] 1. A method of treating a neuroendocrine disorder in an
individual, the method comprising the step of administering to the
individual in need thereof a therapeutically effective amount of a
composition including a TEM, wherein administration of the composition
reduces a symptom of the neuroendocrine disorder, thereby treating the
individual. [0107] 2. A use of a TEM in the manufacturing a medicament
for treating a neuroendocrine disorder in an individual in need thereof.
[0108] 3. A use of a TEM in the treatment of a neuroendocrine disorder in
an individual in need thereof. [0109] 4. A method of treating a
neuroendocrine disorder in an individual, the method comprising the step
of administering to the individual in need thereof a therapeutically
effective amount of a composition including a Clostridial neurotoxin and
a TEM, wherein administration of the composition reduces a symptom of the
neuroendocrine disorder, thereby treating the individual. [0110] 5. A use
of a Clostridial neurotoxin and a TEM in the manufacturing a medicament
for treating a neuroendocrine disorder in an individual in need thereof.
[0111] 6. A use of a Clostridial neurotoxin and a TEM in the treatment of
a neuroendocrine disorder in an individual in need thereof. [0112] 7. The
embodiments of 1 to 6, wherein the TEM comprises a linear
amino-to-carboxyl single polypeptide order of 1) a Clostridial toxin
enzymatic domain, a Clostridial toxin translocation domain, a targeting
domain, 2) a Clostridial toxin enzymatic domain, a targeting domain, a
Clostridial toxin translocation domain, 3) a targeting domain, a
Clostridial toxin translocation domain, and a Clostridial toxin enzymatic
domain, 4) a targeting domain, a Clostridial toxin enzymatic domain, a
Clostridial toxin translocation domain, 5) a Clostridial toxin
translocation domain, a Clostridial toxin enzymatic domain and a
targeting domain, or 6) a Clostridial toxin translocation domain, a
targeting domain and a Clostridial toxin enzymatic domain. [0113] 8. The
embodiments of 1 to 6, wherein the TEM comprises a linear
amino-to-carboxyl single polypeptide order of 1) a Clostridial toxin
enzymatic domain, an exogenous protease cleavage site, a Clostridial
toxin translocation domain, a targeting domain, 2) a Clostridial toxin
enzymatic domain, an exogenous protease cleavage site, a targeting
domain, a Clostridial toxin translocation domain, 3) a targeting domain,
a Clostridial toxin translocation domain, an exogenous protease cleavage
site and a Clostridial toxin enzymatic domain, 4) a targeting domain, a
Clostridial toxin enzymatic domain, an exogenous protease cleavage site,
a Clostridial toxin translocation domain, 5) a Clostridial toxin
translocation domain, an exogenous protease cleavage site, a Clostridial
toxin enzymatic domain and a targeting domain, or 6) a Clostridial toxin
translocation domain, an exogenous protease cleavage site, a targeting
domain and a Clostridial toxin enzymatic domain. [0114] 9. The
embodiments of 1 to 8, wherein the Clostridial toxin translocation domain
is a BoNT/A translocation domain, a BoNT/B translocation domain, a
BoNT/C1 translocation domain, a BoNT/D translocation domain, a BoNT/E
translocation domain, a BoNT/F translocation domain, a BoNT/G
translocation domain, a TeNT translocation domain, a BaNT translocation
domain, or a BuNT translocation domain. [0115] 10. The embodiments of 1
to 9, wherein the Clostridial toxin enzymatic domain is a BoNT/A
enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain,
a BoNT/D enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic
domain, a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT
enzymatic domain, or a BuNT enzymatic domain. [0116] 11. The embodiments
of 1 to 10, wherein the targeting domain is a sensory neuron targeting
domain, a sympathetic neuron targeting domain, or a parasympathetic
neuron targeting domain. [0117] 12. The embodiments of 1 to 10, wherein
the targeting domain is an opioid peptide targeting domain, a galanin
peptide targeting domain, a PAR peptide targeting domain, a somatostatin
peptide targeting domain, a neurotensin peptide targeting domain, a SLURP
peptide targeting domain, an angiotensin peptide targeting domain, a
tachykinin peptide targeting domain, a Neuropeptide Y related peptide
targeting domain, a kinin peptide targeting domain, a melanocortin
peptide targeting domain, or a granin peptide targeting domain, a
glucagon like hormone peptide targeting domain, a secretin peptide
targeting domain, a pituitary adenylate cyclase activating peptide
(PACAP) peptide targeting domain, a growth hormone-releasing hormone
(GHRH) peptide targeting domain, a vasoactive intestinal peptide (VIP)
peptide targeting domain, a gastric inhibitory peptide (GIP) peptide
targeting domain, a calcitonin peptide targeting domain, a visceral gut
peptide targeting domain, a neurotrophin peptide targeting domain, a head
activator (HA) peptide, a glial cell line-derived neurotrophic factor
(GDNF) family of ligands (GFL) peptide targeting domain, a RF-amide
related peptide (RFRP) peptide targeting domain, a neurohormone peptide
targeting domain, or a neuroregulatory cytokine peptide targeting domain,
an interleukin (IL) targeting domain, vascular endothelial growth factor
(VEGF) targeting domain, an insulin-like growth factor (IGF) targeting
domain, an epidermal growth factor (EGF) targeting domain, a
Transformation Growth Factor-β (TGFβ) targeting domain, a Bone
Morphogenetic Protein (BMP) targeting domain, a Growth and
Differentiation Factor (GDF) targeting domain, an activin targeting
domain, or a Fibroblast Growth Factor (FGF) targeting domain, or a
Platelet-Derived Growth Factor (PDGF) targeting domain. [0118] 13. The
embodiments of 8 to 12, wherein the exogenous protease cleavage site is a
plant papain cleavage site, an insect papain cleavage site, a crustacian
papain cleavage site, an enterokinase cleavage site, a human rhinovirus
3C protease cleavage site, a human enterovirus 3C protease cleavage site,
a tobacco etch virus protease cleavage site, a Tobacco Vein Mottling
Virus cleavage site, a subtilisin cleavage site, a hydroxylamine cleavage
site, or a Caspase 3 cleavage site. [0119] 14. The embodiments of 1 to
13, wherein the Clostridial neurotoxin is a BoNT/A, a BoNT/B, a BoNT/C1,
a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, a BuNT, or any
combination thereof. [0120] 15. The embodiments of 1 to 16, wherein the
TEM is administered to an Arnold's nerve or a nerve from the recurrent
laryngeal nerve complex.

EXAMPLES

[0121] The following non-limiting examples are provided for illustrative
purposes only in order to facilitate a more complete understanding of
representative embodiments now contemplated. These examples should not be
construed to limit any of the embodiments described in the present
specification, including those pertaining to the compounds, compositions,
methods or uses of treating a neuroendocrine disorder.

Example 1

Treating a Panic Disorder

[0122] A woman complains of chest pains and dizziness due to her
high-stress job. After routine history and physical examination, a
physician diagnosis the patient with a panic disorder involving abnormal
sympathetic/parasympathetic neuron and identifies the nerves and/or
muscles involved in the condition. The woman is treated by injection of a
composition comprising a TEM as disclosed in the present specification,
targeting the nerves stimulating the hypothalamus. Alternatively, the
woman may be treated by injecting a composition comprising a TEM and a
suboptimal amount of a BoNT/A as disclosed in the present specification.
The patient's condition is monitored and after about 1 week from
treatment, the woman indicates she has decreased chest pain and dizziness
and can properly perform his work duties. At two and four month
check-ups, the woman indicates that she is still experiencing decreased
panic episodes. This decrease in panic episodes indicates a successful
treatment with the composition comprising a TEM and a BoNT/A as disclosed
in the present specification.

[0123] A similar therapeutic effect can be achieved with a suboptimal
amount of any of the Clostridial toxins disclosed herein.

Example 2

Treating a Phobic Disorder

[0124] A man complains of having unreasonable fear of small places and
avoids places like elevators at all costs. After routine history and
physical examination, a physician diagnosis the patient with a phobic
disorder involving abnormal sympathetic/parasympathetic neuron activity
and identifies the nerves and/or muscles involved in the condition. The
man is treated by injection of a composition comprising a TEM as
disclosed in the present specification, targeting the nerves stimulating
the hypothalamus. Alternatively, the man may be treated by injecting a
composition comprising a TEM and a suboptimal amount of a BoNT/A as
disclosed in the present specification. The patient's condition is
monitored and after about 2 weeks from treatment, the man indicates he
has decreased anxiety and can now use the elevator at work. At two and
four month check-ups, the man indicates that he is still experiencing
decreased fear of small places. This decreased in fear indicates a
successful treatment with the composition comprising a TEM.

[0125] A similar therapeutic effect can be achieved with a suboptimal
amount of any of the Clostridial toxins disclosed herein.

CONCLUSION

[0126] In closing, it is to be understood that although aspects of the
present specification are highlighted by referring to specific
embodiments, one skilled in the art will readily appreciate that these
disclosed embodiments are only illustrative of the principles of the
subject matter disclosed herein. Therefore, it should be understood that
the disclosed subject matter is in no way limited to a particular
methodology, protocol, and/or reagent, etc., described herein. As such,
various modifications or changes to or alternative configurations of the
disclosed subject matter can be made in accordance with the teachings
herein without departing from the spirit of the present specification.
Lastly, the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope of
the present invention, which is defined solely by the claims.
Accordingly, the present invention is not limited to that precisely as
shown and described.

[0127] Certain embodiments of the present invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments will
become apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventor expects skilled artisans to employ
such variations as appropriate, and the inventors intend for the present
invention to be practiced otherwise than specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described
embodiments in all possible variations thereof is encompassed by the
invention unless otherwise indicated herein or otherwise clearly
contradicted by context.

[0128] Groupings of alternative embodiments, elements, or steps of the
present invention are not to be construed as limitations. Each group
member may be referred to and claimed individually or in any combination
with other group members disclosed herein. It is anticipated that one or
more members of a group may be included in, or deleted from, a group for
reasons of convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group as
modified thus fulfilling the written description of all Markush groups
used in the appended claims.

[0129] Unless otherwise indicated, all numbers expressing a
characteristic, item, quantity, parameter, property, term, and so forth
used in the present specification and claims are to be understood as
being modified in all instances by the term "about." As used herein, the
term "about" means that the characteristic, item, quantity, parameter,
property, or term so qualified encompasses a range of plus or minus ten
percent above and below the value of the stated characteristic, item,
quantity, parameter, property, or term. Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the specification and
attached claims are approximations that may vary. At the very least, and
not as an attempt to limit the application of the doctrine of equivalents
to the scope of the claims, each numerical indication should at least be
construed in light of the number of reported significant digits and by
applying ordinary rounding techniques. Notwithstanding that the numerical
ranges and values setting forth the broad scope of the invention are
approximations, the numerical ranges and values set forth in the specific
examples are reported as precisely as possible. Any numerical range or
value, however, inherently contains certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements. Recitation of numerical ranges of values herein is merely
intended to serve as a shorthand method of referring individually to each
separate numerical value falling within the range. Unless otherwise
indicated herein, each individual value of a numerical range is
incorporated into the present specification as if it were individually
recited herein.

[0130] The terms "a," "an," "the" and similar referents used in the
context of describing the present invention (especially in the context of
the following claims) are to be construed to cover both the singular and
the plural, unless otherwise indicated herein or clearly contradicted by
context. All methods described herein can be performed in any suitable
order unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language (e.g.,
"such as") provided herein is intended merely to better illuminate the
present invention and does not pose a limitation on the scope of the
invention otherwise claimed. No language in the present specification
should be construed as indicating any non-claimed element essential to
the practice of the invention.

[0131] Specific embodiments disclosed herein may be further limited in the
claims using consisting of or consisting essentially of language. When
used in the claims, whether as filed or added per amendment, the
transition term "consisting of" excludes any element, step, or ingredient
not specified in the claims. The transition term "consisting essentially
of" limits the scope of a claim to the specified materials or steps and
those that do not materially affect the basic and novel
characteristic(s). Embodiments of the present invention so claimed are
inherently or expressly described and enabled herein.

[0132] All patents, patent publications, and other publications referenced
and identified in the present specification are individually and
expressly incorporated herein by reference in their entirety for the
purpose of describing and disclosing, for example, the compositions and
methodologies described in such publications that might be used in
connection with the present invention. These publications are provided
solely for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an admission
that the inventors are not entitled to antedate such disclosure by virtue
of prior invention or for any other reason. All statements as to the date
or representation as to the contents of these documents is based on the
information available to the applicants and does not constitute any
admission as to the correctness of the dates or contents of these
documents.